"

From 2012hs.igem.org

We'll need to take further steps to create finished yeast expression vectors before we can collect data and draw conclusions. However, some lessons that we've learned from this process are:

1. It makes sense to invest a lot of effort planning a large-scale project like this so that the system you're developing has a better chance to succeed.

2. Performing pilot experiments to compare several methods is a good investment of time so that you can learn early what works best.

3. Cloning doesn't always work the first time. You may need to try more than once, adjusting your strategy based on previous results.

4. It's important to label your tubes carefully and save them in an organized way in case you need to go back to previous steps at a later date.

Conclusions about cloning strategies for this project

1. There's a fairly high background of colonies without inserts when doing blunt cloning; this can be improved by optimizing the insert:vector ratio for the ligations. This method has the advantage of only requiring one DNA cleanup. Blue/white selection helped with screening, but this is an additional expense and could be detrimental to the cloning process, particularly for protein-coding sequences.

2. The sticky XmaI strategy seemed to better for obtaining colonies with inserts and seemed to have a lower background (less of a need for blue/white selection). The disadvantages are of course that the insert can't have an XmaI site and that this method requires 2 DNA cleanups. However, it was the method that was successful for obtaining the most problematic clones, so including the XmaI sites (or something comparable) in the tails may be a helpful strategy in the long run.

3. Using blue/white selection may be helpful for promoters and terminators, but it may be good to avoid it for protein-coding sequences in case their expression is toxic.

Next steps

1. Obtain a yeast cloning vector with appropriate BsaI sites as well as a terminator clone.

2. Make clones of a promoter, a fluorescent protein coding sequence, and a terminator in the yeast cloning vector. Transform these plasmids into yeast and quantify promoter activity based on expression of the fluorescent protein.

3. Create complete genes that can be used in future Advanced Biotechnology and Molecular Biology classes to monitor biological processes.

Return to our main page.