Build-a-BUG: Series 2, Session 1
Saturday, February 9, 2013
Noon to 4:00 PM
This is the first Build-A-BUG workshop in a series of five on yeast. Students are introduced to basic lab techniques, including pipetting and centrifugation, and start their own mating type detector projects. This lab involves crash courses on molecular and cellular biology, the life cycle of budding yeast, and BioBrick-based approaches to synthetic biology, as well as hands-on minipreps (DNA isolation).
- We did minipreps on overnight E. coli cultures containing BioBrick parts BBa_K801000 (pTUM100) and BBa_K165057 (Kozak + mCherry). These minipreps were performed using both the commercial QIAGEN QuickLyse kit and the published One-Step protocol. The DNA was eluted in distilled H2O and stored at -20°C.
- We designed our yeast mating type detector construct. We will use BioBrick Assembly standard 10. First, we will ligate the inducible promoter (either BBa_K110006 or BBa_K110005) to the translational unit (either BBa_K165057 or BBa_K165059) to the terminator (BBa_J63002). After a quick PCR verification (to ensure that the promoter was not directly ligated to the terminator), we will ligate this assembly into the vector (BBa_K801000). See the whiteboard assembly strategy images below for a visual representation.
- Caveat: According to the Parts Registry, the promoters BBa_K110006 and BBa_K110005 are identical but with the "suffix and prefix switched (right to left)." We are not exactly sure what this means so Tom PCR amplified both parts. If time permits, we will sequence them and use BLAST (Basic Local Alignment Search Tool) to select the functional promoter for our project. Otherwise, we will make constructs with both promoters and see what happens.
- We looked at the agarose gel that Tom ran with the PCR products of BioBrick parts BBa_K110006, BBa_K110005, BBa_K165059, and BBa_J63002.
- We feasted on charcoal-grilled burgers!
Yeast life cycle and sporulation
QuickLyse and One-Step miniprep protocols
Assembly strategy (pre-cut)
Assembly strategy (post-cut)