BUGSS:Build-a-BUG:2-1: Difference between revisions
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* 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 H<sub>2</sub>O and stored at -20°C. | * 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 H<sub>2</sub>O 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. | * 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 [http://www.yeastgenome.org/cgi-bin/blast-sgd.pl 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. | ** ''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 [http://www.yeastgenome.org/cgi-bin/blast-sgd.pl 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 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! | * We feasted on charcoal-grilled burgers! | ||
Latest revision as of 09:51, 18 February 2013
Home Contact Internal Lab Members Projects Protocols Build-a-BUG Build-a-Gene 2016 Build-a-Gene 2015 Build-a-Gene 2014 Build-a-Gene 2013 Chalk Talks
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).
Background reading
- A Primer on Molecular Biology: Section 1.1 (pp. 1-17) provides a concise introduction for the non-biologist.
- Life Cycle of the Budding Yeast Saccharomyces cerevisiae
- Sporulation in the Budding Yeast Saccharomyces cerevisiae
- An Introduction to BioBricks
- BioBrick Assembly standard 10
Protocols
BioBrick parts
- BBa_K801000: pTUM100 yeast shuttle vector based on pYES2
- BBa_K110006: Alpha-Cell Promoter MF(ALPHA)1
- BBa_K110005: Alpha-Cell Promoter MF(ALPHA)2
- BBa_K165057: Kozak + mCherry translational unit
- BBa_K165059: Kozak + CFPx2 translational unit
- BBa_J63002: ADH1 terminator from S. cerevisiae
Notes
- 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!
Whiteboard
