Significant gene set testing - limma
The mroast function has an argument to specify which contrast do you want to test, quoting from the help page:


  contrast
  contrast for which the test is required. Can be an integer specifying a column of design, or the name of a column of design, or a numeric contrast vector of length equal to the number of columns of design.


So if you have the contrast of 1-min timepoint positive vs 1-min timepoint negative you can use this.
Read the help pages in detail, specially the help pages in limma are quite extensive and detailed to answer most questions if not all about how to use the software.