Assign each person in your group a different plasmid to analyze.
You will first need to assemble the complete sequences of your overexpression plasmids by combining the plasmid and MET gene sequences. Recall that the S. cerevisiae genes have been cloned into the pBG1805 vector and that the S. pombe genes and LacZ have been cloned into the pYES2.1 vector. You will then generate a restriction map that can be used to predict restriction fragments generated in an RE digestion. To generate the map, we will use one of several online tools that is available at the website of New England Biolabs, a commercial supplier of REs.
Use the table below to calculate the length of your plasmid as you complete the first few steps of this exercise.
1. Locate the coding sequence of your gene.
- Open the record for your MET gene or its homolog at SGD or Pombase, respectively.
- Find the number of amino acids in your protein. Multiply this number by 3 to find the number of nucleotides in the CDS. Add this number to the table above.
- Find the CDS sequence for your gene. You will paste this sequence to the end of the plasmid sequence in step 2.
- The pYES2.1- lacZ sequence is posted on Canvas, so steps 1 and 2 are already done for this plasmid. Note that plasmid lacZ gene has been modified from naturally-occurring lacZgenes.
2. Assemble the complete nucleotide sequence of your plasmid.
- Open the Word document containing the pYES2.1 vector sequence posted on Canvas. The plasmid sequences are numbered so that the GAL1 promoter is at the 3’-end of the DNA sequence.
- Copy the CDS from SGD or Pombase and paste it at the end of the plasmid sequence.
- Delete the last three nucleotides of the CDS, which comprise the gene’s stop codon. The overexpression plasmids are designed to encode fusion proteins with C-terminal exten- sions. (Note: the stop codon in the lacZ sequence has been removed by the manufacturer.)
3. Prepare a restriction map of the complete plasmid sequence.
- Paste the sequence from step 2 into the search box in the NEBCutter tool: nc2.neb.com/NEBcutter2/
- Check the box to indicate that the plasmid is CIRCULAR, rather than linear.
- You might also want to give your plasmid a name. The NEB site will store your queries for 24 hours, which can be very convenient. Click submit.
- The search tool will return results for a bewildering number of REs. The vast majority of the RE sites are not useful, because the fragments are too large or too small, the enzyme is not available in the lab, or the endonuclease is sensitive to DNA methylation (which can be unpredictable).
4. Perform custom digests with enzymes that look promising.
- Click the custom digest link. This brings up a chart of REs that cut the plasmid, their recognition sites, the number of recognition sites, and the amount of enzyme activity in
each of four buffers.
- Analyze the fragments that would be produced by the four REs below by checking the box and clicking the green Submit button.
AccI HincII ScaI XbaI
(Note: There may be some changes to this list of available REs before the lab.)
5. Prepare a table summarizing the restriction maps for your three plasmids.
- Complete the table below, indicating the sizes of the restriction fragments generated witheach RE.
- Include the total length of the plasmid in the table. The sum of the restriction fragment lengths should sum up to this number.
6. Choose a RE that distinguishes your three plasmids.
The team should use the data table above to select the RE that best allows you to distinguish the three plasmids. We will be analyzing the restriction fragments on 1% agarose gels, which do a good job of resolving fragments ranging in size from ~500 bp to ~5000bp. Refer to the figure in Chapter 8, which shows the distribution of molecular size markers on 1% agarose gels. Choose an RE that will produce restriction digests with a nice range of fragment sizes.