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Team:BroadRun-NorthernVA/Notebook/Weeks 1and2
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Lab Notebook
Welcome to our Lab Notebook! Here, we have documented the work done in our project so we can see and keep track of how our project is progressing.
June
This year, we decided to start an IGEM team for our local High School- Broad Run. Previous attempts in previous years were unsuccessful because we had trouble finding a sponsor and students to join. However, this year we found both a sponsor as well as multiple students with different backgrounds to help us engineer genes. Since we are Broad Run's first IGEM team in history, we hope to make IGEM bigger and better in our community so that our school can stay competitive in such a distinguished competition.August 8
- Designed 3 amylase gene constructs to be synthesized through IDT’s offer. The amylase gene used for all three constructs was the alpha amylase coding sequence from Bacillus amyloliquefaciens. The Genbank accession number is J01542.1. First parts used were pCyc (medium) promoter (Part BBa_I766555), Kozak sequence (Part BBa_K165002), Alpha amylase coding sequence (Genbank Accession #J01542.1), and ADH1 Terminator (Part BBa_K801012).The alpha amylase coding sequence contained an EcoR1 restriction site, so GeneDesign was used to eliminate the site and optimize the construct for S.cerevisiae.
However with this combination, IDT would not accept our design, due to repeats and sections with a low GC count. The constructs were redesigned, keeping the same coding sequence and Kozak sequence, but using the minimal cyc promoter (Part ) and minimal adh1 terminator (Part ). In order to test several variants on the expression of amylase, two promoters, and two secretion sequences were used. Plasma DNA was used to map restriction sites, and Gene design used to remove restriction sites. The final makeup of the gene constructs are listed below.
Construct 1
- Biobrick prefix
- Promoterless
- Kozak sequence (Part BBa_K165002)
- Native secretion sequence, from Bacillus amyloliquefaciens
- Alpha amylase coding sequence from Bacillus amyloliquefaciens
- ADH1 Terminator (Part BBa_K392003)
- Biobrick Suffix
Construct 2
- Biobrick prefix
- Minimal cyc promoter (Part BBa_K105027)
- Kozak sequence (Part BBa_K165002)
- Native secretion sequence, from Bacillus amyloliquefaciens
- Alpha amylase coding sequence from Bacillus amyloliquefaciens
- ADH1 Terminator (Part BBa_K392003)
- Biobrick Suffix
Construct 3
- Biobrick prefix
- Minimal cyc promoter (Part BBa_K105027)
- Kozak sequence (Part BBa_K165002)
- Native secretion sequence, from Bacillus amyloliquefaciens
- Alpha amylase coding sequence from Bacillus amyloliquefaciens
- ADH1 Terminator (Part BBa_K392003)
- Biobrick Suffix
