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IGEM:IMPERIAL/2006/The Talk Page
- 1 Purpose of this Page
- 2 A message to Tom and John S.
- 3 Major Issues (Everyone please read and contribute)
- 4 General Issues
- 5 Resolved Issues
- 6 Random Ideas
- 7 Working Bank Holiday Weekend
Purpose of this Page
Tom is away revising for exams and John is going away to the US. This page is a place where questions are raised and answered and a place to keep everyone aware of what is going on. Also, if anyone external to the project wishes to raise a point, contribute to a discussion or simply to ask a question please feel free!
A message to Tom and John S.
Christin 20:08, 6 September 2006 (EDT): Since labwork has become quite heavy and we have been feeling the limit in manpower since you two left - could you help us out doing some data analysis, revising of protocols & extracting parameters etc. After Friday, we will be down to 4 people and then soon next week there will only be 3 people be available to work ... so every help that can be done via the wiki or similar is urgently needed! Here are some of the most important things for now:
- Revising & checking protocols:
- J37015 has been revised - can it be checked and amended if necessary - it is being tested in the lab now
- J37022 protocol needs to be checked & revised - hopefully there will be a construct ready on Thursday to be tested
- Analysis of data obtained so far:
- For now, J37016 has given quite good results - and we have a good set of data that can be used & has to be analysed more thoroughly now. Data is up on the general results page or on the T9002 experimental data page
- Lots of data to come the next days (hopefully)
If you are unsure about anything & where the project is proceeding (from just looking at the wiki) - let us know immediately. Also, if you find any flaw / mistake - give us a notice asap so we can still have a chance to fix any errors.
- Johnsy 21:03, 6 September 2006 (EDT): I'll try to have a look at the data for J37016 and J37020 within the next few days. I have posted a note on the general results page.
- Johnsy 21:23, 6 September 2006 (EDT): I have altered the first three sections of the J37022 protocol. Have we gotten the ELISA plates and the biosensor working yet? Have we gotten any decent results for the J37015 prey cell yet and what is happening to the CreLox part and the Riboswitch construct? Are they working as well? Keep me updated... Also, if anyone has any questions for Dr. Endy or anyone here at MIT, I might be able to meet with him to get an answer.
- JohnChattaway 04:41, 7 September 2006 (EDT) We are no longer using elisas, we are using a type of western blot which uses the imunotags.
- Johnsy 09:09, 7 September 2006 (EDT): In that case, can you please update the J37022 protocol page to reflect that. Thanks! (Or is there another protocol page that has been made for J37022?)
- Farah 12:48, 7 September 2006 (EDT)': Hi John. I'm currently updating that protocol now. The biosensor has (sadly) become a low priority in our 'to do' list. The CreLox part is a really difficult PCR and hasn't shown any promise so far (Johnny has tried to make it work many many times) so we've left it for Kirsten to have a go at if she has any time. We've more or less given up on the riboswitch. As an alternative method of controlling our positive feedback loop, we've decided to just add acylase to the medium to get rid of any AHL. We're currently testing how our acylase affects growth of the E.coli. If it works then it's probably the simplest way to control the feedback loop. As for the J37022, I am reading over your protocol for testing the half-life of aiia and I am unsure about the second half. From what I understand from your comments, both parts of the protocol seem to be testing the same thing but the second half requires a three hour wait, the reason for which I don't understand. Can you clarify please?
- Johnsy 15:21, 7 September 2006 (EDT): Yes, indeed the two parts of the protocol measure the exact same thing. What we were trying to do is to get the half-life from two distinct experimental methods and to see if they correlate in any way. This could prove to be useful in the future, and it doesn't take any more time, as you still have to wait for AiiA expression to reach a high enough level for it to be measured. However, if you don't feel comfortable with using that part of the protocol, it can just as easily be left out. The only thing I'm worried about is the western blot technique. Will this give us a quantitative value for the half-life of AiiA? I'm asking since I'm not too familiar with the protocol for western blot.
- Farah 13:56, 7 September 2006 (EDT): This is for John & Tom. We may discard the idea of testing for half-life of aiia anymore since it's stable and so we can say the half-life is equal to cell-division. Also, since we are using the chemostat, we are able to control cell growth and so we can tweak with the half-life of the enzyme. What we really need to know are the k2 and Km of aiia...can you guys do some quick digging and find these values?
- Tom 14:10, 7 September 2006 (EDT): Check the realistic values page. It should be there.
- Johnsy 15:31, 7 September 2006 (EDT): I have to disagree with the idea of discarding measuring the half-life. We still need to be able to compare the half-life with the average cycle period we expect. If the half-life is longer than the cycle period we expect, we won't be producing any oscillations. Moreover, the AiiA is using a degradation tag which will speed up the degradation of the AiiA protein. I'm unsure of how this will affect the half-life of the AiiA. Also, if we assume that the half-life is equal to the time needed for cell division, then we are assuming that the concentration halves every 20 minutes, which is untrue if we are considering the total population wide concentration. In fact, the concentration will tend to double every 20 minutes as the cells begin to metabolise and make more AiiA. BUT...this is all dependent upon how good the chemostat is at maintaining cell population. If it is very efficient, then I guess you could assume that the AiiA would halve every 20 minutes (assuming that the rate at which AiiA is removed outweighs the actual rate that AiiA is degraded...which I'm not sure if this is a good assumption to make until we get actual values of the half-life of the AiiA we are using.
- Farah 07:43, 14 September 2006 (EDT):From what I know of the chemostat, it is quite efficient and so we should be able to make the assumption that the half-life of AiiA is equal to the division time. The problem is, it's the eleventh week and we still don't have the AiiA test construct to be able to determine the half-life. It turns out that the terminator we were using for the construct and the final predator actually isn't there (according to Davidson) so we're trying the ligation with a different terminators from the registry this time. Dr. Mann offered to continue ligations of the test constructs and the final predator if we're available for testing during term time.
- Johnsy 22:46, 14 September 2006 (EDT): My class has a matlab tutorial on how to use it to calculate the least square curve fit, so I will get back to you next monday on the values hopefully...
- JohnChattaway 16:39, 23 September 2006 (EDT) It is true that the halflife will equil the division time but the division time is dependent on the flow rate of the chemostat so it will vary between 1-2 hours aprox.
Major Issues (Everyone please read and contribute)
Cre Lox
Non-functional AiiA (Unresolved)
- John C: Data showing aiiA does not work
- Tom 13:55, 26 August 2006 (EDT): I'd be a little hesitant to condem AiiA with that data alone, for the following reasons:
- The control of having non-induced cells I don't think is adequate. Having non-induced cells as a control we are assuming the operator sequence is functioning and intact and we're also making the assumption there is enough native LacI repressor to effectively shut off AiiA production. We need a control where we have AHL innoculated with non-AiiA/LuxI/Flourescence producing cells.
- Secondly, the method for working out whether the difference between induced and non-induced cells is statistically significant is not given. Just because a difference is between 3-4% doesn't make it statistically insignificant by default. Especially if you're consistently getting those results. I'd suggest a T test on repeated samples to determine significance.
- Thirdly, the dilution of cells and small AHL innoculation time may be having an effect. I'd suggest growing a culture overnight in the prescence of AHL and IPTG, along with appropriate controls, and taking the supernatent directly from that for the T9002 assay.
- Johnsy 06:49, 27 August 2006 (EDT): I agree with Tom in that the AiiA may work. There are several reasons for non-expression (or maybe non-detection?) I have not tried to increased the IPTG amount, so maybe there is not enough IPTG to induce expression. Moreover, as our T9002 has been acting up within the past few days, it might be our T9002 cell activity which is the problem. However, having run an SDS gel with no results might signify that there is not expression of AiiA in the first place (or that there is not enough expression to produce any significant results). We might want to try to increase the IPTG concentration so that we are sure to induce AiiA expression and retry the SDS gel? just a suggestion...Then again, it could be the problem with the part itself in the registry. We can wait for another few days until our J37022 test construct finishes and try to run the tests using that construct.
- Tom 19:14, 27 August 2006 (EDT): I reckon first thing to do would be to induce overnight in the prescence of AHL. You can then do the T9002 assay straight from the o/n culture, and use some of the cells for the SDS page.
- JohnChattaway 07:53, 28 August 2006 (EDT) I think you may be right, the western blot expriment is flawed because DH5a can't express protein at high levels, to do that you need to use a different strain so the western blot may have failed for that reason. The new strain kirstin wants to use is in the next safety catagory above DH5a but that just means it has more forms to be fille in. Secondly I think that seeing as the activity of aiia is so low an hour may not be a long enough time period to get mesurable results I will try an assay which runs overnight and look into small chemostats to run it in.
- Tom 10:00, 28 August 2006 (EDT): I don't think a chemostat would be necessary. Just whack in some IPTG and AHL in a standard overnight culture. Just to act as a diagnostic as to whether AiiA is being produced and is working. You shouldn't need a chemostat until assembley of the final system is complete.
- JohnChattaway 04:48, 29 August 2006 (EDT) The reason no aiia is being produced is that we were testing a contaminant. The part should grow in kanomycin media only but we were testing in ampacillin so it was an amp-resistant contaminant growing, not the real part. will retest in kan medium
- Tom 06:27, 29 August 2006 (EDT): The AHL assay test data should be okay though, as I'm pretty sure John S tested in Kanamycin (I set up a culture with Deepti, defiantley used Kan). And as we were only doing the western blot because we didn't think that was working, doing the overnight innoculation/induction is still probably the best move.
- Johnsy 18:00, 29 August 2006 (EDT): Yes indeed I have been using Kanamycin for S01656 testing. Furthermore, I believe that we grew up the S01656 culture for SDS gel in Kanamycin, so there should still be some expression of AiiA. However, the culture might be contaminated already, since when we were growing up the cultures, we accidentally grew up in Amp, but we did have substantial bacterial growth :S...not really a good sign...and might be a contaminant of something else...but definitely not good. I would susupect that maybe the AiiA genome might have been mutated or as John C suspects, that the AiiA isn't being produced enough in the medium, so we are not getting enough expression to be measured.
- Tom 13:54, 31 August 2006 (EDT): What's going on with this? I saw in the lab-notebook that S01656 was meant to have been tested today but no mention of any results? Plus, presuming the S01656 testing hasn't been done, we still don't know if DH5a are producing AiiA yet (as an overnight inoculation still hasn't been done), so why are we transforiming BL21 cells under the pretense that DH5a isn't producing protein?
- Tom 13:59, 4 September 2006 (EDT): Has the overnight been done yet? Sorry to go on about it because from what's being reported in the lab book, it appears you've all but given up on the AiiA with, what I can see, no extra data then what you had a week ago.
- Christin 04:17, 5 September 2006 (EDT): No, S01656 has not been given up yet. It could not be tested yesterday because we did not have any T9002 culture that worked. This is another problem - T9002 does not give us any good results.... we think this might be due to a mutation in the cells (before they were frozen) because it seems that none of the frozen cells seem to give good results. T9002 has now been electroporated anew straight from the registry such that it can be tested tomorrow and hopefully, we will get better results. S01656 is going to be tested again today - I think there might have been a problem as well with the T9002 assay at that time (if you compare the data obtained for one day), so that the problem might not even be the S01656. As we know the J37016 is working, I will try to use that one to check for AHL degradation. This time, an overnight inoculation was done as well. There will be some data coming tonight ....
- Johnsy 15:18, 5 September 2006 (EDT): Are we still updating the protocols continuously? I haven't seen any updates on them since last week :S...
- The final constructs were sequenced and there was no aiiA It looks like it was the registary's fualt JohnChattaway 06:53, 13 September 2006 (EDT)
General Issues
Finalised Predator Construct
- J37018 or J37019???
- Monocistronic finalised prey cell or Poly cistronic prey cell?? (2 promotors and single promotor respectively).
- We have to decide soon guys!!! If all goes well, we may be making this baby by the end of next week!!
- Tom 14:03, 4 September 2006 (EDT): My vote is for using the single promoter. Although that's just off preference rather than any data. Has anyone been analysing the results from the testing? PS. I thought one of the predators was J37020?
- Christin 04:35, 5 September 2006 (EDT): I am in favour of the single promoter too. My reason rather is that J37020 does NOT give any good results from the testing whereas J37016 (polycistronic predator construct) gives very good results. The actual polycistronic predator has been finished ligating and can be checked tomorrow...
anyone thought of using an auxostat rather than a chemostat? (just to make it even more complicated)
- Johnsy 17:52, 4 September 2006 (EDT): What is an auxostat?..pls. clarify...
- Tom 18:55, 26 August 2006 (EDT): You'll all be pleased to know it is possible to assess AHL levels via HPLC. They did it here. Dunno whether you can incorporate this into testing, but I defiantley think it's a more accurate way to assess AHL levels.
- Johnsy 07:11, 27 August 2006 (EDT): Again, the problem with using HPLC is that we will not have a direct measurement of the AHL levels in the actual oscillator. This will be okay for our testing phase, and actually might be helpful if we begin by testing the concentrations of the AHL in the stock AHL, so we are sure that we are using correct AHL concentrations. Degradation of the AHL (or incorrect concentrations) might account for some of the bloopers we have had in our T9002/J37016/J37020 testing.
- Tom 09:16, 27 August 2006 (EDT): There is facility in the bioreactor suite to take samples from the chemostat and run them through a HPLC within the room. There is no reason we can't use this to test AHL levels in the oscilator, although I think the GFP flourescence would be a nicer reporter. It could, however, prove useful if things don't go according to plan and we have to troubleshoot. And if you think the AHL is messed up, it might be an idea to make up a new stock when Vincent gets back.
- Farah 18:27, 28 August 2006 (EDT): John, you've landed!! And you're already typing on the wiki! Just have to say - thanks for leaving your lab book...As for the HPLC - I think we should go ahead and do it so that we can check our T9002 transfer curve but to save manpower and time I'd rather give it to the ABC to sort out than do it for ourselves.
- Johnsy 18:02, 29 August 2006 (EDT): Will the other stuff in the LB medium and the bacteria cell affect the HPLC readings, or do we have to purify AHL somehow?
- Tom 14:06, 4 September 2006 (EDT): Unsure on this, all depends on the resolution of HPLC really. Which I don't know. Can anyone find this out? Plus if purification was needed you might be able to run some kind of hydrophobic precipitation, but again, unsure on the details.
Protein Half-Lives
- Tom 12:04, 24 August 2006 (EDT): Hi guys, something to note which I don't think has been that clear. In a growing cell culture cells grow and divide, increasing the volume of cells. If the production of a stable protein was shut off then the effective half-life of that protein would be the division time of the cells. IE, as the cell becomes two, the protein is shared between a volume twice as large as the original. Thus halving concentration. This needs to be taken into account when assessing protein half-lives.
- Although note, this does not effect AHL half-life as its volume is not constrained by the cell, but rather the fixed volume of the medium.
- Christin 12:51, 24 August 2006 (EDT): After cell division, the protein concentration would half - that means that that protein concentration is not in steady state any more but after a certain time, the steady state concentration would be reestablished again. How can this be taken into account?
Also, this time delay might have effects on the dynamics of the system.
- Tom 10:16, 25 August 2006 (EDT): You would still get establishment of steady states when protein is being produced. The only thing cell division is effecting is protein half-lives when we consider the volume of cells in the chemostat is always the same. IE, the washing out rate is the same as the growth rate. And thinking about it, in the chemostat AHL half-life would also be equivalent to the doubling time of Bacteria, IE ~30 minutes, as this is being washed out at the same rate. PS: Is the full model on the wiki yet?
- Johnsy 07:18, 27 August 2006 (EDT): Something else to note regarding the cell division: Once the cell divides, yes the concentration of AiiA is halved WITHIN each cell, but eventually, the AiiA concentration will again reach a steady state and return to the level before, which means that in time, there would be an actual increase in the amount of AiiA within the entire system. However, if we consider the prey cells, they too are dividing. Thus, also increasing the concentration of AHL (doubling it every doubling time). Intuitively, this would suggest that our amplitude would double every time the cells divide (which is not necessarily a bad thing?), but more modelling needs to be done on this to confirm.
- Tom 11:23, 27 August 2006 (EDT): The changing of amplitudes won't happen within the chemostat, as the number of cells remains constant. And the actual division of cells themselves doesn't effect protein levels in the way that there is a sudden drop and increase back to steady state. The act of division itself does not effect concentrations as the resulting daughter cells will still have the combined volume of the single parental cell. What is effecting concentrations is growth, which is continuous.
- Christin 19:11, 29 August 2006 (EDT): Wait a second ... the concentration within the cell as it divides does not change at all. As concentration is number of moles per volume, the concentration stays the same. However, are you saying that the growth of the cell before it divides will affect the concentrations? Yes, it does, but is there such a large change in cell size that it will have a significant effect on the concentrations?
- JohnChattaway 09:26, 1 September 2006 (EDT) I hate to point out the obvious but the cells grow by increacing their size so this can be ignored by keeping the cells OD constant in a chemostat.
- JohnChattaway 04:52, 7 September 2006 (EDT) The halflife of AHL in the chemostat will be affected by washout in exactly the same way as any intracellular protein. This is because media + cells is removed so as far as the chemostat is concerened there is no diference between the two areas of the media. Secondly the argument that a constant OD will cause any intracellular protein to remain constant while an extracellular protein can be washed out is flawed as there is a constant amount of empty media inside the chemostat! so they are both constant.
Monitoring Cell Populations
- Tom 07:35, 21 August 2006 (EDT): Hi everyone, just been reading through this. Big drawback of the 3 antibiotic way of monitoring cell population that you've identified is the extra ligation steps. What extra ligations steps are involved? If you just mean grafting your parts into a plasmid with appropriate antibiotic markers, can't you just co-transform a seperate plasmid with the marker on, thus saving a ligation step? I'm pretty sure those kind of plasmids would be easy to get hold of as they are the basis of a lot of gene cloning.
- Tom 10:03, 21 August 2006 (EDT): Actually, on second thoughts, that wouldn't work, as you'll end up losing the plasmid containing the antibiotic you're not screening for while the cells are growing
- Johnsy 11:30, 21 August 2006 (EDT): From what I understand from the team, we will use the antibiotic way of monitoring as a backup plan, in case we decide to monitor cell populations. For now, we have not decided to monitor cell populations, but we will go ahead and electroporate & Maxi the parts we might need. Just in case...
- Deepti 15.24, 22 August 2006 (EDT) -Talking to Dr. Jensen yesterday morning, it seems that the 3 antibiotic method is not very accurate an requires extra ligations steps which can be avoided.
- Waiting for plated samples to grow will require atleast a day before we can assess the differences in population sizes.
- The number of bacteria in the plated samples does not necessarily accurately reflect the relative proportion in the common broth culture.
- Even if the plating method were to be an accurate measure of the colony sizes in the broth culture, we could still only record, and not control population sizes
- According to Dr. Jensen, the best way to do it would be the OD method.
- By growing up the bacteria separately, we can measure their separate growth rates which, hopefully, should not be too disimilar.
- When mixing the two cultures, the ODs should be equal, and preferably, between 0.6 and 0.8.
- Tom 13:05, 22 August 2006 (EDT): Would it be possible to check the population ratios after they've been mixed together using the OD method?
- Deepti 09.10, 23 August 2006 - I'm afraid not. Once the cultures are mixed there is no way to tell them apart using the OD method. Best we can do, I reckon, is ensure the OD of both individual cultures is between (0.6-0.8) right before mixing and then just hope they stay more or less equal (we will have a rough estimate of the individual growth rates of each population from the OD measurement of the individual cultures done beforehand).
- Tom 05:27, 23 August 2006 (EDT): If we're gonna be using the chemostat for the final assembley (which I assume we are), Dr. Leak made it clear that it amplifies the smallest difference in compeitive growth ability. Meaning that once innoculated, there's a good chance the ratios are gonna change. I strongly recommend looking more into this, and looking into obtaining LacZ plasmids.
- JohnChattaway 09:24, 1 September 2006 (EDT) Dr david leak made it clear to me that this was the least of our problems
Measuring LuxI
- Farah 12:22, 21 August 2006 (EDT): What are we doing about luxI in J37015? Are we going ahead with measuring the GFP levels in that part? Tom mentioned talking to Vincent about it...I can model the luxI but from the assumptions we've made for our current model for J37015 we ought to get a similar curve as for the AHL levels. To Tom: I remember you saying you've talked to Vincent about the luxI - what was the conclusion?
- Tom 15:50, 21 August 2006 (EDT): Yeah, we decided it might be easier and more accurate to measure the GFP from the pLux transcript as opposed to AHL. We'd still like to measure AHL to see the relationship between LuxI and AHL, however the relationship may be easier to characterise and more accurate than the T9002 assay.
- Deepti 23 August 2006: What would be the point of measuring LuxI if we cannot characterise the relationship between LuxI concentration and AHL concentration? The only way we could do that is to measure both compounds (using both protocols) and come up with a curve that describes their relationship. Correct me if I am wrong, but as far as I can gather, the reason for using this alternative protocol is to achieve greater accuracy than with the AHL assay. Using the alternative protocol we would gain a more accurate measurememnt of LuxI concentration but the AHL measurement would still depend on the original assay whether we use the curve produced by the alternative or not!! (Since the curve is extracted by comparing data from both assays).
Should we not stick with the original assay then, inaccurate as this may be? Unless ofcourse we do want to measure the concentration of LuxI in the system, for which I'm not sure I see the reason for.
LuxR in the Predator Cell
- Tom 15:50, 21 August 2006 (EDT): I remember when Farah and Christin assumed LuxR in the predator was constant we still got oscillations. If this is the case what is the point of LuxR being under the control of pLux as opposed to a constitutive promoter? Also, if this is the case and LuxR variance isn't important, than our design is exactly the same as the preivious MIT design, except spread over two cells. Maybe someone can look at the effects on the system of having LuxR vairable and LuxR constant?
- Farah 11:29, 22 August 2006 (EDT): When Christin and I assumed LuxR to be constant we were just 'hiding' the fact that LuxR is actually varying - as was pointed out by Vincent. I don't see the poing in modelling a simplified version of the system when our more 'accurate' model is working.
- Tom 13:39, 22 August 2006 (EDT): I think it would just be interesting to see the differences. We've made LuxR vary, and if we still get oscillations when it isn't varied people may ask why we bothered to make LuxR variable in the first place.
- Farah 18:20, 28 August 2006 (EDT): Sorry I didn't answer you sooner about this - just noticed your comments buried in the mass of text on this page. We don't really have the option of choosing whether levels of LuxR vary or are constant. Our predator (J37016 or J37020) just simply has varying levels of LuxR, so assuming constant LuxR levels in the modelling would just hide what was actually going on in our system (we tried it but this point was pointed out to us by Vincent). If we adapted the predator to produce constant LuxR then we could make the approximation but this could only be done with J37020 which has two promoters - doing so for part J37016 would mean producing constant amounts of AiiA too.
- Tom 13:12, 13 September 2006 (EDT): Sorry if I was unclear. I wasn't suggesting to actually make the thing, but just to model it. Just so we can see whether having LuxR fluctuate gives us an advantage. For example, MIT did this without fluctuating LuxR, and its very possible they'll ask us our rationale for making it variable. And if we don't know what varied LuxR does as opposed to constant LuxR then we won't be able to answer that question.
Cre/Lox Part
- Johnsy 07:08, 27 August 2006 (EDT): What is going on with the Cre/Lox part? Have we started to ligate it yet?
- Jonny 12:59, 27 August 2006 (EDT) This is being done by Ms. Jensen and now I think Dr. Mann also. Basically, we have to do two seperate PCR reactions and then fuse them together. One of the PCR reactions appears to be fine, however the other has failed four times. The reason for the failures is thought to be the very long overhanging fragment for one of the primers - around 60 base pairs. They have been playing around with the different temperatures, but there has been no success so far. If it continues to fail, then an option is to order two seperate sequences that anneal to each other which should then make the PCR simpler. Unfortunately none of the other ligations steps are possible without the PCRed fragment.
- Johnsy 18:04, 29 August 2006 (EDT): Ok...cool...but if it doesn't work, is it a very necessary part to have for our prey cell? Have we finished testing the J37015? or rather..how is the testing going for the J37015?
- JohnChattaway 09:44, 12 September 2006 (EDT) The cre lox part now exists and we may test it this week
Resolved Issues
Prey cell positive feedback test
- JohnChattaway 05:55, 22 August 2006 (EDT): I am curious about what data this test will actually give us. It seems like you are measuring the time taken to reach steady state rather than characterising the positive feedback loop. I think that a much better test would be;
- Make a fresh day culture of J37015 (OD 0.4 (guess))
- Make a T9002 AHL assay
- then every 10 mins for 2hrs
- Take 1 1ml sample
- Record OD
- Centrifuge down cells, collect supernatant
- Put supernatant on ice
- After 2 hours this should leave you with 12 samples of different AHL conc in the ice bucket you can test these with the T9002 assay.
- We would then normalise the AHL reading against OD
- Tom 07:07, 22 August 2006 (EDT): That's pretty much exactly what the protocol says to do. Although it needs to be adapted to look at GFP output over time also. Also it'll have to be made more clear if you had trouble working out what was going on, any volunteers? :D
Non-functional Prey Cells
- John C: The prey cells don't flouressce! This is a majour problem!!! what are we doing about it?
- This data suggests that the prey cells are flourescing however there is no propper controll so the experiment needs to be repeated. I am pretty sure the increaced flourescence is due to the presence of cells not producing GFP. There needs to be a positive controll.
- My experiment yesterday showed that the prey cells DO NOT PRODUCE AHL! this is a big problem.
- How can we fix this?
- Tom 14:10, 26 August 2006 (EDT): When I did the initial testing of J37015 I initally got a AHL and flourescence flatline. However, repeating the experiment and missing out the dilution steps from the o/n culture I got this output for AHL (although I forgot to measure J37015 direct flouresence, oops!). The data does show that AHL is being produced.
- Tom 10:01, 28 August 2006 (EDT): Any updates on this?
- JohnChattaway 04:40, 30 August 2006 (EDT) I have decided that the prey cells are producing AHL based on tom's data. Here's how I think we can test the prey cell positive feedback loop, uising a chemostat.[1]
Random Ideas
Jamboree Demonstration
- Tom 18:43, 26 August 2006 (EDT): Hi guys, just thought I'd pop in with an idea. We were saying how we wouldn't be able to have a live demonstration at the jamboree because our period is too long. However, what about a simulated live demonstration? So we have a computer generated visual representation of the culture running off our model, and we can change parameters like cell ratios, growth rates and chemostat ODs. That way people can come along and fiddle with the paramters and be like 'wow, I can chage frequency/amplitude really easily!'. Plus, as it'll be based off a mathematical model, it isn't gonna go wrong in front of them. What do you reckon? I personally think audience participation is always a big plus. Also, just generally and not thinking about the jamboree, it'll be interesting and potentially very useful to see how the model responds to these new input parameters that we can easily change.
- baijiongjun: hi, i'm finally back at china, still have limited access to internet, will be back in action soon, how's the project going on? how's everyone, will be pop in more in the wiki soon.
- Johnsy 18:04, 4 September 2006 (EDT): As I am currently updating this webpage from the Stata Center (where the iGEM jamboree will be held - quite a funky building indeed!), I am thinking of our presentation, as it is already the 10th week. I apologise for not visiting the site within the past few days. My aunt uses a dial up connection that is too slow to load web pages at any reasonable speed, and accessing the internet would mean that her landline was cut off whilst surfing the web. But finally, I have access to MIT's incredible wifi system which works just about everywhere on campus :)...I was thinking of the following sections to add into our presentation:
- Introduction
- What is an oscillator?
- What is the motivation behind a biological oscillator?
- What has been done in the past? - but of course not to step on anyone's toes
- Design
- How our design is different to previous designs (MIT & Repressilator)
- The funky stuff we though of to control our oscillator, including the riboswitch, Cre/Lox system, chemostat
- Our quest for the holy AiiA...and Vincent's chemostat
- Modelling
- Our mathematical models for the entire system
- I don't think we need to go into detail about the models for the individual testing constructs..we can just say that we obtained the values...as like MIT did in their 2004 IAP presentation
- Results/Implementation
- After all that testing, the results we obtained from our testing constructs
- Did our final assembly work in the end? if not, can we explain our errors?
- Video of working system? or at least a nice animation of what it is supposed to look like?
- Conclusion
- What have we learned to do and not to do with synthetic biology?
- Is synthetic biology worth pursuing in the future?
- Future developments with our biological oscillator
- Analysis of our project, suggestions for future iGEM teams wishing to tackle the problem of the biological oscillator.
- Any other suggestions?
- Introduction
- JohnChattaway 09:47, 12 September 2006 (EDT) I think we should focus on how our AHL system is based on predator prey dynamics because it is actually quite a complicated concept which will take 10min to explain fully, this will generate more interest in our project than all the little tricks we developed to alter how it works
- Johnsy 08:50, 13 September 2006 (EDT): We also have a poster presentation to do. I think we should try to keep the presentation as simple as possible and have all the details in the poster presentation. This would prevent our presentations from getting too boged down and from preventing people from falling asleep. Yes, we do need to go into some details, which is okay, but too much details might have the reverse effect of what we want.
Working Bank Holiday Weekend
- Farah 18:37, 27 August 2006 (EDT): This is to John. Can you come in tomorrow afternoon to read the plates and culture up stuff for tuesday? I can call/mail you to let you know when you need to come in. We've worked it out so that then you, Christin, Jimmy and I will only need to contribute a half day...Our fate lies in your hands....
- Farah 17:51, 28 August 2006 (EDT): We've gathered a lot of data this weekend. Could everyone process whatever data they have (using the spreadsheet)and update the results page?
- Johnsy 18:05, 29 August 2006 (EDT): Pity they didn't have a holiday here in the US...but we do have one next Monday 4 Sept! :) Good to see you guys still dedicated!


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DescriptionChatbots are one of the most exciting and in-demand topics in tech, with businesses around the world exploring their potential for cutting costs and improving customer service. You can capitalize on the demand and kickstart your own career in this booming field with this professional certificate program from IBM. Jump in, and you'll learn how to build useful chatbots for businesses and deploy them to their sites. You'll explore creating a business out of chatbot building and emerge with the know-how to help businesses leverage chatbots in their operations. Plus with this offer, you'll get one year of Watson Assistant services to power ten chatbots at no charge.
InstructorFounded by Harvard University and MIT in 2012, edX is an online learning destination and MOOC provider, offering high-quality courses from the world's best universities and institutions to learners everywhere.
Important Details

subset: commoncrawl


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In 1969, educator Dr. Laurence J. Peter and Canadian playwright Raymond Hull wrote The Peter Principle, which famously stated that "In a hierarchy every employee tends to rise to his level of incompetence … in time every post tends to be occupied by an employee who is incompetent to carry out its duties … Work is accomplished by those employees who have not yet reached their level of incompetence."
That's a pretty disheartening thought for advisors focused on growing their business for the future, yet more than 40 years later, research on leadership transition conducted by the Harvard Business Review showed 70% of employees lack the critical attributes required for success in future roles. Among high-potential employees moving to new positions within the company, 40 % of those moves end in failure. In essence, most companies have tools and processes to identify talent but they lack a process for ensuring the right talent gets moved into higher-level jobs. Instead, they may "reward" their best performers by giving them higher-level positions, unintentionally moving them out of the role that best suits them.
Talent management could be considered the antidote to the infamous Peter Principle. Talent management is a formalized process used to identify and facilitate development of employees and leaders for the future of your practice. Talent management guides you in assessing the strengths and development needs of your employees and identifying employees with high potential for future professional and leadership roles within your practice. Through talent management, you create an action plan for developing the competencies that will be critical to those roles, which improves the ability of your practice to meet your business objectives.
Harvard Business Review's research identified the critical attributes for future success:
- Ability: The intellectual, technical, and emotional skills to handle increasingly complex challenges
- Engagement: The level of personal connection and commitment employees feel towards firm and its mission
- Aspiration: The desire for recognition, advancement and future rewards and the degree to which the employee's wants align with what the company wants for him/her
Failure to properly consider these attributes sets up your employees for failure, costs your business time and money and creates disengagement and disillusionment on the part of the employee who has been miscast. As you go through the TA process, keep in mind the purpose: assessing talent and development needs; identifying employees with high potential for future assignments; and building succession plans for critical roles within the practice.
A talent management plan for your business starts with a talent assessment, i.e., assessing the talent you need to grow your business. You will be developing that talent over time, so your assessment should take a long-term view: what skill sets and competencies are needed to successfully meet your business goals and initiatives?
The next step is to review your current staff and determine in which competencies they have strengths and where there are gaps. Then decide what actions can be taken to close the gaps: additional education or training, mentoring, motivation or even hiring. Create a talent development plan for each of your existing employees. If you identify gaps which may require a new hire, create a job description that includes the critical competencies for that role.
Your employees are one of your greatest assets. If you are not developing their talent and skills, that asset is depreciating. Take the initiative now to start planning for the practice you want in the future.


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It was the year before prime minister Indira Gandhi was assassinated (1984). The nation was not in the best of moods. There were raised voices and frayed tempers prevalent across the nation as corruption and intolerance reached an all-time high. Given the milieu "Jaane Bhi Do Yaaro" (JBDY) chose to laugh away the bruise. There is a sharp zaniness about JBDY, which serves as a scathing antidote to the growing sense of collective dismay that the nation faced as extremism, and inflation hit the nation hurling it towards damnation.
Many regard JBDY as the funniest Hindi film ever made. I am not too sure. A sense of deep sombreness behind the giggles and guffaws. The plot about two out-of-work photographers named, hold your legal notices, Vinod Chopra (Naseruddin Shah) and Sudhir Mishra (Ravi Baswani), has episodes of acute hilarity and interludes of absolute anarchy.
You know from the Faustian flavour, the Shakespearean fervour and frenetic pacing, that there was an abundance of improvisation on the sets. You also know as you watch the film`s newly restored edition that the film was made on a shoestring budget, which afforded meager finesse, and practically no re-takes. Nope. Nothing can restore the austerity and begging-bowl crisis that the film had to face.
The actors had to literally survive by their instincts, which could only take the narration this far and no further. There are too many references and cross-references to politicians and scams.
The nexus between politics and journalism is brought out through a character named Shobha Sen (Bhakti Bharve) a hard-nosed newspaper editor who uses our two heroes to get an expose on a builder named Tarneja (Pankaj Kapur) and his murky collaboration with a municipal officer named D`Mello (Satish Shah).
D`Mello soon ends up as a corpse in a traveling coffin that leaves us chortling heartily or coughing uneasily, depending on how far we are willing to accept the opulent oeuvre of the outlandish and the outrageous that the film throws forward.
In one sequence we see the dead-drunk builder Ahuja (Om Puri) driving into the coffin carrying the dead D`Mello thinking it`s another vehicle. The sequence is funny only if you are hell bent on enjoying the goofiness of a grand high-school reunion fete bringing together grownup professionals who decide to "have fun" for one evening even if some of their actions make them look downright silly.
In JDBY everyone is out to have fun. It`s a very boys kind of nudge-nudge-wink-wink fun where the brunt of the joke is the system that fosters and encourages corruption on every level. The characters discuss malpractices without shame or purdah. The cast is mainly masculine. The two women in the cast Bhakti Bharve and Neena Gupta behave like the boys.
Bharve`s hard-nosed journalist`s act is astonishingly unladylike for those times. She uses her "charms" to outwit her male adversaries and to make Vinod putty in her hands. In the end, when Sudhir and Vinod are framed for scams, which they had set out to expose, Shobha walks away from the duo leaving them to face the music. There is an unmentionable `b` word for such exploitative women.
Among its many pioneering achievements is the use of the inspirational song "Hum honge kaamyaab", a desi rendering of the song "We shall overcome", a protest song conceived for the African-American Civil Rights Movement. It went on to acquire a renewed popularity through its ironic usage in the film.
To this day "Jaane Bhi Do Yaaro" remains a crazy film, filled with madcap situations straight out of comedies from the silent era with a corpse rolling down Mumbai`s highways and time-bomb`s planted to kill the "good" blowing up in evil faces.
The message is loud and clear: farce can fight a moral-political fascism.
Characters talk incessantly either about corruption or about being corrupt. We can see the downslide in the moral values in Indian politics and bureaucracy was already happening in a major way.
Yes, Amitabh Bachchan had much reason to be angry. The Big B`s anti-establishment film "Andha Kanoon" rubbed shoulders with Govind Nihalani`s "Ardh Satya" during the same year that Kundan Shah`s comedy of `eras` made an impact.
Significantly this was also the year when the pre-Bachchan superstar Rajesh Khanna had his last spate of hits - "Avtaar", "Souten" and "Agar Tum Na Hote". Romance in cinema was dying. The angry brand of heroism was being favoured. "Jaane Bhi Do Yaaro" is also an angry film, but the wrath of the common man is here seen in a mirthful mood. Laugher is taken to be the best medicine to fight injustice.
The film rightly celebrated climactic confusion on stage where the film`s characters mingle with the characters from the "Mahabharata" to create a poetic panic and anarchy served well as a metaphor for the tangle that the country had created in its socio-political system months before Mrs. Gandhi was killed.


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Search in all content
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- 1 - 25 out of 1,343 results
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"Developing the Management Studies Community" Grant
Koen van Bommel (Recipient) & C.M.J. Wickert (Recipient), 2018
Prize › Academic
"Best Master Thesis" Award 2013 (€5,000)
Anne-Madeleine Kranzbühler (Recipient), 2013
Prize › Academic
ZonMw Parel voor onderzoek naar preventie van psychose
M. van der Gaag (Recipient), 30 May 2013
Prize › Academic
ZA/DFID Growth and Labour Markets in Low Income Countries Programme (GLM/LIC) Grant
R.H. Oostendorp (Recipient), 2014
Prize › Academic
Young Talent Prize - Dutch Neuroscience Meeting
B.R. (Barbara) Braams (Recipient), 2018
Prize › Academic
Young Scientist Outstanding Poster Prize
Martijn Westhoff (Recipient), 2008
Prize › Academic
Women's empowerment, social norms and domestic violence
W. Janssens (Recipient), P. Collier (Recipient), M. Groot Bruinderink (Recipient) & K. Morsink (Recipient), 1 Dec 2016
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Winner of the StudenTalent Prize 2017 of the Faculty of Social Sciences, VU Amsterdam
Maria Charlotte Rast (Recipient), 3 Jul 2017
Prize › Academic
Winner of the Regional Science Association Internanational Dissertation Competition
Eric Pels (Recipient), 2000
Prize › Academic


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Article content
OTTAWA - Liberal Leader Justin Trudeau has a new definition of Canada's middle class that could exclude anyone who has some money socked away for a rainy day.
"For me, it's people who live paycheque to paycheque," Trudeau said Wednesday.
That's a switch from what the Liberal leader said earlier in the week.
"People who live off their incomes are the middle class and those who live off their assets, their portfolios, their trust funds are not," Trudeau said Tuesday, similar to how he defined middle class a day earlier.
When Trudeau raised the issue of middle class debt and financial stress in the Commons during question period, Prime Minister Stephen Harper tore into Trudeau's new paycheque-to-paycheque definition of middle class.
"So that would exclude, Mr. Speaker, people of modest incomes who have saved something, like pensioners, who are not living from paycheque-to-paycheque," he said. "And, of course, Mr. Speaker, it would include people who have a very affluent lifestyle and spend all their money even though they get a very large paycheque."
The prime minister said Trudeau's comments leave him confused.
"I have no idea what the leader of the Liberal Party is talking about and I suspect he doesn't either," he said.
Trudeau shot back that Harper needed "to spend more time with middle class Canadians."
Harper responded with more ridicule for Trudeau.
"If the leader of the Liberal Party was with middle class Canadians he wouldn't actually know whether they were middle class or not," he said.
Trudeau's shifting definitions come as he tries to make fighting for the middle class the Liberals' hallmark issue for next year's federal election campaign.
"We are focused on helping middle class Canadians and helping Canadians who hope to achieve middle class status," he said.
Trudeau has promised not to raise taxes on the middle class, but he has yet to define which tax brackets or income levels would be safe from any tax hikes under a Liberal government.
Trudeau said Wednesday he'd "let economists argue about which quintile or decile (the middle class)" starts or ends at.
Carleton University economist Ian Lee took up the challenge.
He said Trudeau's latest definition of middle class is meaningless because it's "a psychological, subjective test."
Instead, Lee said he defines the middle class by excluding the richest and poorest Canadians from his calculations.
"The top 20%, they're the rich people," he said. "The bottom 20% are the really poor people, you know, people on welfare, people in social housing."
Lee said that leaves "the middle 60% of Canadians" as the middle class.
He also insists the group is easily defined.
"It's an empirical, measurable amount of income coming in to you," he said.
The latest Statistics Canada data for families of two or more people in Lee's definition of middle class had annual incomes ranging from $43,000 to $132,000 in 2011.
Lee adds that wide range captures the full gamut of middle class, including what he calls the lower and upper middle classes.
tap here to see other videos from our team.


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Diabetes is a chronic disease that occurs when the pancreas is no longer able to make insulin, or when the body cannot make good use of the insulin it produces.
Insulin is a hormone made by the pancreas, that acts like a key to let glucose from the food we eat pass from the blood stream into the cells in the body to produce energy. All carbohydrate foods are broken down into glucose in the blood. Insulin helps glucose get into the cells.
Not being able to produce insulin or use it effectively leads to raised glucose levels in the blood (known as hyperglycaemia). Over the long-term high glucose levels are associated with damage to the body and failure of various organs and tissues.
There are three main types of diabetes:
Type 1 diabetes used to be called juvenile-onset diabetes. It is usually caused by an auto-immune reaction where the body's defence system attacks the cells that produce insulin. The reason this occurs is not fully understood. People with type 1 diabetes produce very little or no insulin. The disease may affect people of any age, but usually develops in children or young adults. People with this form of diabetes need injections of insulin every day in order to control the levels of glucose in their blood. If people with type 1 diabetes do not have access to insulin, they will die.
Type 2 diabetes used to be called non-insulin dependent diabetes or adult-onset diabetes, and accounts for at least 90% of all cases of diabetes. It is characterised by insulin resistance and relative insulin deficiency, either or both of which may be present at the time diabetes is diagnosed. The diagnosis of type 2 diabetes can occur at any age. Type 2 diabetes may remain undetected for many years and the diagnosis is often made when a complication appears or a routine blood or urine glucose test is done. It is often, but not always, associated with overweight or obesity, which itself can cause insulin resistance and lead to high blood glucose levels. People with type 2 diabetes can often initially manage their condition through exercise and diet. However, over time most people will require oral drugs and or insulin.
Symptoms of type 1 diabetes
Symptoms of type 2 diabetes
Both type 1 and type 2 diabetes are serious. There is no such thing as mild diabetes.
Gestational diabetes (GDM) is a form of diabetes consisting of high blood glucose levels during pregnancy. It develops in one in 25 pregnancies worldwide and is associated with complications to both mother and baby. GDM usually disappears after pregnancy but women with GDM and their children are at an increased risk of developing type 2 diabetes later in life. Approximately half of women with a history of GDM go on to develop type 2 diabetes within five to ten years after delivery.
Other specific types of diabetes also exist.


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Videos | August 10, 2012
Mars Curiosity Rover Update (Aug. 10, 2012)
Mars Science Laboratory team member Jessica Samuels gives a progress report on Curiosity's first days on Mars.
Transcript:Hi, I'm Jessica Samuels, member of the Engineering Operations Team, and this is your Curiosity rover report.
So since the nail-biting entry, descent and landing event, we're happy to report that Curiosity is healthy and clicking off all the activities that we asked her to perform for the first few days on the surface.
We first deployed the high gain antenna to facilitate communications back to Earth so that we can communicate with Curiosity much more efficiently. We've deployed the remote sensing mast so we can start acquiring all our beautiful panoramic images. And we've also checked out what the state of the rover is by looking at ourselves in addition to the Martian landscape. We were happy to find that we weren't covered in a large amount of dust as some members of the team though might happen. In actuality, we just have some of these pebbles that have strewn across the rover deck with just a fine, fine layer of dust, which is well within the operating limits of the rover.
So coming up we're going to give the rover a brain transplant. We're wiping away all of the cruise and entry, descent and landing software and making room for the software needed to perform the exciting portions of the surface mission ahead. We're giving the rover the capability to do the sampling portion of the mission as well as being able to drive more efficiently to different targets on the surface. While we're doing the software upgrade, we'll still be sending images from the entry, descent and landing event, as well as different images from the panoramas that will give us a view of what like around the rover.
So that's your Curiosity rover report. Please check in often for future updates.


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- Minimum 500 PIC passenger flying hours on type and 1,500 passenger flying hours.
1. Updated CV.
2. Valid Pilot License.
3. Valid Medical Certificate.
4. Passport copies.
5. Last two pages of logbook.
6. Personal photo.
7. Clearance from license authority for no accident or incident involvement.
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Raquette Lake seeks resolution
Adirondack Daily Enterprise
November 2, 2013
By MIKE LYNCH
RAQUETTE LAKE - It's rare for an issue in the Adirondacks to have unanimous support of environmental groups, local governments and state lawmakers in Albany, but that's the case with Proposition 4, a constitutional amendment that will be on the Election Day ballot Tuesday.
Proposition 4, like any constitutional amendment, had to be approved by two separately elected state Legislatures and then submitted for voter approval statewide.
Those who endorse Proposition 4 include the Adirondack Association of Towns and Villages, Adirondack Landowners Association, Hamilton County Board of Supervisors, Long Lake Town Council, Adirondack Council, Adirondack Mountain Club, Adirondack Wild, Protect the Adirondacks, the Open Space Institute, New York Snowmobilers Association, former Gov. George Pataki and the owners of the contested property, among others.
If passed by voters, the proposition would amend Article 14, the "forever wild" section of the state constitution. The proposition aims to solve a land dispute between the state and private parties in Raquette Lake, a tiny community in rural Hamilton County, that goes back to the 1870s.
In Raquette Lake, 216 parcels consisting of about 1,034 acres have contested titles. The state claims the land is Forest Preserve while private entities, many of them with deeds, claim to own the land. These properties contain private homes, businesses, a school building, a town waste transfer station, a private marina and even a firehouse.
The proposed amendment would allow the Legislature to settle the dispute by working a deal between the state and the property owners. The Legislature would give up the state's claim to the properties in exchange for additional Forest Preserve that would be paid for by the private entities. Companion legislation that outlines the process was already passed in both houses this past summer.
One of the contested properties
It is widely expected that the state Department of Environmental Conservation would target the Marion River Carry as the property to add to the Forest Preserve, although the DEC has said it's premature to assume this will be the parcel. The canoe carry, which consists of 295 acres, has been coveted by
environmentalists and paddlers who want to ensure that it remains open to the public. The land is currently owned by the Open Space Institute, which purchased it from Raquette Lake resident Dean Pohl earlier this year.
The Raquette Lake property owners would be expected to pay $2,000 each, plus an additional amount based on their property's assessment. The total expected to be raised is about $600,000. That money would be held by the town of Long Lake, which would use it to purchase the land.
"This is clearly, I would say, the best way to resolve the issues, given the fact that Article 14 and the constitution doesn't allow for resolution other than litigation or constitutional amendment," said Long Lake Supervisor Clark Seaman, whose town includes the hamlet of Raquette Lake. "Litigation is incredibly expensive for both parties, and this really does seem to be a win-win for all parties involved."
A long history
The contested land that is located on the shores of Raquette Lake was originally part of Township 40, which consisted of 24,000 acres and was part of the Totten and Crossfield Purchase in the 18th century. Back then, surveyors divided the Adirondacks into 50 areas, called townships, so land could be easily sold.
The state claims it purchased the properties from private owners in 19th-century tax sales and that they became part of the Adirondack Forest Preserve. Residents and the other parties have disputed that, saying they own their property legally. The attribute the title dispute to inadequate surveying, poor record keeping, clerical errors and lost documents. Many of the property owners hold deeds and note that a number of those tax sales that allowed the state to acquire property were ruled illegal by the courts.
The state made numerous attempts in the early 20th century to claim the lands as Forest Preserve. In some instances, they took residents to court, and other times, they tried to get them to sign over their property. Often, the property owners stood their ground.
"The defendants claim the right to the property they occupy by virtue of deeds obtained from the people who formerly occupied these several sites," stated a New York Times article from Nov. 15, 1903, about the state's effort to claim the land at the time. "They claim the state's title to the land, which was obtained by virtue of tax sales, is defective, in that the land was sold as non-resident land, when, as a matter of fact, it was owned and occupied by the defendants."
Today, the residents and state still each claim the land, but both agree the compromise that is outlined in Proposition 4 is the best solution.
Passing Proposition 4 would end this same debate that caused headaches for state workers and was traumatic at times for those living under the cloud of the title issue. Not having clear title has affected people's and organization's ability to make improvements to their structures, subdivide their lots and take out mortgages, among other things. Plus, residents have lived under the threat that someday the state Attorney General's Office would take them to court and try to take away property that they pay taxes on and has been in private hands for more than a century.
However, the property isn't treated as Forest Preserve by the state most of the time. Most of it is developed and off limits to the public because it is occupied by private homes or other structures. Even the state Adirondack Park Agency labels the contested lands as private on its land-use map.
Raquette Lake property owner Carolyn Gerdin has done a tremendous amount of research on the title dispute and has represented the property owners in many discussions with government officials and environmental groups. She said relations are now strong with state officials, but she acknowledges there have been problems in the past that have dragged this issue out over time.
Gerdin has experienced some of these problems firsthand. She said her father bought the family's property on Raquette Lake in 1950. At the time, he checked to see if the land had a legitimate title and believed it did. A former owner had even gotten permission to file deeds with the state in the 1930s. However, her dad also noticed the state considered the land to be contested.
"So he called DEC and he asked them what their attitude toward these occupancies was going to be, and they assured him ... that they were working on and expected to have an equitable solution imminently," Gerdin said. "That was 65 years ago this November."
Her family's contested title didn't become an issue until 20 years ago, when the family was subdividing the lot after her dad died. Gerdin said she got an APA permit for the subdivision and was in the process of getting permits for an underwater cable to bring electricity to the property, which is standard on the lake. That got the attention of the state, she said, and the question of her contested title was turned over to the Attorney General's Office.
"That's when I sat up, took notice and said, 'Oh, boy. We're in for it,' and I began to research our property and trace the deeds back," she said.
Although Gerdin's family was concerned about what the Attorney General's Office might do at the time, they haven't heard anything since about it.
"It's an emotional thing to go through this," said longtime Raquette Lake resident Jim Blanchard, who has been interviewed by the Enterprise numerous times in recent years. Blanchard lives full-time in Raquette Lake and has a deed to his property that goes back to 1896, yet the land is still contested.
Blanchard's family, which has lived on Raquette Lake for five generations, actually won a court case against the state to clear its title to another property on the lake in the 1950s. For the most part, residents have been successful when taking the state to court to clear their titles. Property owners have beaten the state in eight of 10 cases the Enterprise is aware of. Most of those were before the 1950s.
Despite this success rate, those interviewed by the Enterprise said they don't want to go to court because it's extremely time-consuming and expensive.
"What happened is that all the people that cleared their title were second home owners up here," Gerdin said, referring to early 20th-century lawsuits. "The poor guy that's been living up here since 1840, and there were some, were not the people who could afford these attorneys."
Neil Woodworth, an attorney and the executive director of the Adirondack Mountain Club, said the case history of the state losing is one reason that his organization would like this issue resolved through the constitutional amendment.
"I also looked at many of the rest of the claims, and frankly, the legal background on property titles to most of these properties is so twisted that I had little confidence that a state Supreme Court justice could unravel this, because of insufficient evidence," he said. "So (the constitutional amendment) seemed to be the best course, since the state was clearly not going to commit and force people off their properties even if it won the case. You know, we just don't do that in the 21st century."
The DEC is very guarded with what it says about the title issue because it is not allowed to take stances on constitutional amendments, but documents obtained by the Enterprise through the Freedom of Information Law show that it has been frustrated by this issue in the past.
One letter that expresses this sentiment was written by former DEC Region 5 Director Thomas Monroe on Sept. 18, 1990. Monroe acknowledges the "long-standing controversy" and said he dealt it with as far back as the 1960s.
"When I was an Assistant Forest Surveyor working out of the Northville Office in 1964 we had a question arise regarding a boundary in that township whereupon I head up with my survey crew to try and work it out," he wrote. "The next day the District Director of Lands & Forests Maynard Fisk intercepted me at the Northville office and inquired as to my activities and subsequently instructed me to cease and drop any further work in that township.
"It is my long standing belief that the Department and the residents of that area would be well served through having these title questions resolved."
He went on to say he was intrigued by the suggestion of a constitutional amendment to clear title on lands with structures occupying them.
Another letter, by former DEC land surveyor Kenneth Yousey in 1989, also described the situation as tough to deal with from a state worker's perspective.
"Township 40 appears to be an impossible situation," Yousey wrote. "We can only attempt to contain the problem until someone comes up with a solution to it."
Monroe's case of being pulled out of Raquette Lake wasn't the only time a state surveyor was told by a superior to alter their work plans there. Another time was in June 2004, when a DEC worker doing survey work put up Forest Preserve signs in people's backyards.
This actually came during a period when the property owners and state officials were in the early stages of starting to come up with a plan for solving the title dispute.
The signs didn't stay in the yards very long because residents phoned then-Assemblywoman Teresa Sayward, who retired in 2012, to complain. Sayward, Sen. Betty Little and many Hamilton County officials have been supporters of Raquette Lake residents for years and have often lobbied on their behalf.
"I called the commissioner of DEC, and I said, 'Pull your boys back. Get them off this land," Sayward said, recalling the situation. "How are we ever going to resolve an issue if there's antagonism up there?"
Sayward said it turned out the surveyor wasn't trying to antagonize the residents but simply trying to do his job and wasn't aware of the complexity of the situation.
Still, those types of interactions made negotiations difficult at times. There was a lack of trust between the state and the locals.
"It's a feeling and reaction that took years ... to overcome, and worked both ways, quite frankly," Gerdin said. "We certainly had people at DEC who had very unpleasant experiences up there, and therefore were very leery of us, and the people up there, because they had had unpleasant experiences, were leery of the state. I do think one of the reasons it took so long is that it took a long time for people to get comfortable with one another."
The last 10 years also included one failed attempt to get a constitutional amendment on the ballot in 2008. That deal, Gerdin said, was too costly for the property owners, among other things, and eventually fell through.
But now Gerdin said relations between the interested parties are much stronger, and the proposed deal is something that property owners can live with. She is hopeful the proposition will pass and the issue will be resolved, finally, after 140 years.
She said having this resolved will allow residents to sleep better at night knowing they don't have to worry that their land might one day be taken.
"You will have the confidence, you have the belief that you don't have to worry anymore that something that you would do or want to do is going to bring attention to your place and cause the state to sue you - or threaten to sue you, which is almost as bad," Gerdin said. "I think the number-one thing (will be) just the feeling of massive relief and peace. And that the right and just (action) has finally come around


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A recent spate of attacks on new saplings and existing olive groves in the West Bank over the weekend has left Palestinian olive farmers devastated.
On Sunday 11 January, 170 olive trees were chopped down in the village of Yasuf, south of Nablus near the illegal Israeli settlement of Tappuah. The trees were described as "Roman," a local Arabic term that means that they were at least 100 years old. Olive trees have been known to survive for 1000 years or more, so the trees destroyed in Yasuf may have been truly ancient. This attack came after two separate incidents on Friday south of Hebron, where 345 trees were cut down. The first attack came during the day near the village of Yatta, where 45 trees were cut down. Later that night, under the cover of darkness and the stormy conditions, 300 more trees were hacked down near the illegal Israeli settlement of Susiya. Separately, Palestinians have claimed that 50 olive trees were cut down in the Palestinian village of Kafr Yasif near Acre in present day Israel.
This all comes after approximately 5,000 olive saplings were uprooted in separate incidents on New Year's Day. 80 one-year-old plants were uprooted in Al-Khader village on the Hebron-Jerusalem road, and 5,000 newly planted saplings were pulled up in Turmussaya village near Ramallah. These saplings had been planted in December in honour of the late Palestinian Government Minister Ziad Abu Ein, who died during clashes with Israeli soldiers on 10 December during a demonstration in the area. The landowners filed a report with the Palestinian Military Liaison Office, and Israeli sources have indicated that an investigation is being carried out into the New Year's Day incident.
Palestinian officials and local popular resistance committees have pointed the finger for these attacks squarely at the residents of nearby illegal Israeli settlements. The destruction of Palestinian olive groves has in the past been a key feature of Israeli colonialism in the West Bank, as local farmers can be bankrupted by the loss of their harvest, thus hastening their dispossession and paving the way for further land grabs and illegal settlement construction. Settlers rarely face prosecution and are often given armed protection by the Israeli army.
Olive farming and cultivation is a big part of life in Palestine and a major feature of the Palestinian economy, with an estimated 80,000 families in the West Bank relying on the annual harvest as part of their income. As with all aspects of life under occupation, olive cultivation is conducted at the mercy of the Israeli authorities. Palestinian farmers are under constant threat from settler violence and intimidation, and some 800,000 olive trees have been cut down in the occupied West Bank since 1967. Additionally, they also face having their olive groves designated as closed military zones with restricted or even completely forbidden access.
Since the end of the harvest, which took place around October and November, olive farmers will have switched their focus forward. Given that it takes some 5 to 8 years for newly planted olive saplings to bear fruit, olive farmers must be constantly looking to the future to ensure adequate preparation despite deleterious events the likes of which we witnessed last weekend.
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SXSW 2013 Friday review: Green Day, Alt-J, Patty Griffin, Laura Mvula and more
Ricardo Baca
So flip flops were a terrible decision on SXSW 2013's third day. Thankfully, we made plenty of other good decisions on Thursday to balance out the pain of 15 hours on your feet in two-year-old Havaianas.
And it started out by securing tickets for Green Day's intimate show at Austin City Limits Live. The show had the kids all abuzz throughout Austin on Thursday with fluorescent T-shirts flooding the streets and "American Idiot" bumping from their parents' cars. These big shows at SXSW are hardly in the spirit of music discovery, but they're fun for the fans and they bring a certain mainstream legitimacy to the event.
Green Day's set was expectedly familiar with FM hits ("Know Your Enemy") and pop-punk-spirited power ballads ("Boulevard of Broken Dreams"), but it was also full of newer material from the band's trio of new records ("99 Revolutions" kicked the night off). Early on in the concert, frontman Billie Joe Armstrong - fresh from a stint in rehab last fall - brought a young boy to the stage for a feet-first stage leap. Every other song created a sing-along of sorts, prompting Armstrong to quip, "Everything is louder in Texas."
But better than any of the band's songs - from "Burnout," "Welcome to Paradise" or "Holiday" - was the energy the show created. Green Day is used to playing to 15,000-20,000-seat arenas, so seeing them in this 2,700-capacity room was truly a spectacle. They slayed the room, from the ground floor to the balcony, and the kids up front couldn't have been more ecstatic to see their heroes in such a unique room.
Thursday also brought us the best set we've seen so far. British rockers Alt-J threw down a stunning early-evening set at the Mohawk that backed up my call that the Mercury Prize winners' "An Awesome Wave" was the best album of 2012. Hearing that album live was a ridiculous treat, with the record's subtleties captured in a challenging live performance setting.
But they pulled it off. And it sounded better than the record ever will. Playing as a four-piece, they looked like proper English gentleman - but they sounded like the most progressive band in the game. Also: We didn't expect that voice to come from that guy, but when it did, you couldn't help but sing along and throw your hands in the air. When they threw down "Matilda," wow. And when they let out "Breezeblocks" to close the too-short set, the capacity patio fell into a universal groove. (If you're reading this from Denver, it's worth noting that the band's sold-out Bluebird Theater show originally scheduled for April 3 has been moved to April 23, still at the Bluebird. Also on sale, its show at the significantly larger Fillmore Auditorium on Sept. 5.)
The prettiest set of the day came courtesy of New West Records, which threw its day party in a castle of sorts, situated in a residential neighborhood 15 minutes outside of downtown Austin. Songwriter Patty Griffin, new to New West, played a few songs to the packed, pin-drop-quiet room. "Get Ready Marie" had the crowd swaying, and she closed with "I'll Miss You When You're Gone." It was a lovely respite from rock 'n' roll, from bars, from mania. And we felt our heads cleared as we left the party to head back to town.
Others worth mentioning from SXSW Thursday:
Wisconsin-rooted troubadour Trapper Schoepp and his band the Shades played an early slot at the 1100 Warehouse, and even though the crowd was thin, the ever-affable Schoepp threw down a lively set of Midwestern bar rock that was as memorable as it was melodic. He closed with the best song off his "Run, Engine, Run" LP, "Tracks," and that song -- which always reminds me of the Old 97s at their "Too Far to Care" peak -- set the bar high for the rest of the evening.
We caught film director Robert Rodriguez's band Chingon at the Doritos stage. (And that's the strangest sentence I'll write all day.) Rodriguez is competent on guitar, and he was a fierce narrator as he introduced the night's concept: Performing songs to the video backing of the films they appeared in. It worked to varying degrees, but a high point came right away when the band took on its "El Cancion del Mariachi," a song he wrote with Los Lobos for the film "El Mariachi." The mostly-locals audience ate it up.
We stumbled upon DJ/producer Cashmere Cat at Republic Live earlier in the night, and his blend of beats and soul made for an intoxicating mix. He hails from Oslo, Norway, but the kids who filled the dance floor in Texas took him in as one of their own.
British songwriter Laura Mvula gave Griffin a run for prettiest set of the day. Mvula, playing a late-night show at the Hype Hotel, had a startlingly beautiful voice. Sadly the venue was a bit loud, and her full band's sound had difficulty carrying across the warehouse-like space. Still, Mvula's music was what I immediately Spotified when I woke up this morning - and I can't wait to listen to some more after Saturday's fourth and final day of SXSW 2013 wraps up tonight.


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