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Team:WarrenCIndpls IN-HS/Notebook
From 2011.igem.org
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Notebook
May 4th: Bacterial and Yeast Transformation
Bacteria can be made to take in plasmids by adding calcium chloride and heat shocking them.
May 11th: Gibson Assembly
May 19th: Research on Parts
Kozak Sequence- directs translation of mRNA for more efficiency and accuracy; the amount of protein synthesized from mRNA is dependent on the strength of the Kozak sequence
Promoter Region- site for RNA polymerase to attach to and begin transcription; yeast has many promoters within its genome that can be used to express metal detectors
Terminator Sequence- signals the end of transcription to RNA polymerase
Vector- Plasmid
Multiple Cloning Site (MCS)- part of plasmid that can be cut open for genetic modification
Origin of Replication (ORI)- sequence where replication is initiated
Selection Markers
-Ura3 is a selection marker for yeast
-Ampicillin Resistance is a selection marker for bacteria based antibiotic resistance
May 26th: Primer Design
Primers- the 3' section must be complementary to the DNA template, the 5' end may have additional, non-complimentary base sequences to add restriction enzyme sites, and the 3' sections should not be complimentary to each other (increases risk of primer-dimers forming and inhibiting amplification); forward primers extend from start codon to stop codon while reverse primers work from stop codon to start codon
Our Primers:
1st Forward Primer contains an overhang to attach the biobrick to the plasmid, two restriction enzyme sites (for cutting the sequence out), and a primer for the construction of a new strand
2nd Forward Primer contains a primer for constructing a new strand to connect the translational unit to the biobrick
Reverse Primer contains an overhang to attach the biobrick to the multi-cloning site (on the plasmid), the other two restriction enzymes sites, and a primer for constructing a new strand
June 17th: DNA Extraction and Purification
Using detergent, meat tenderizer, and ethanol, we separated the DNA out of the bacteria that contained the m-cherry gene and also the bacteria that contained the ADH-1 terminator.(See project for procedure)Once the DNA separated, we froze it until the following Monday.
June 20th: PCR, Gibson, Bacterial Transformation
Extract out the Prs416 vector from bacteria. PCR the primers and the plasmid to increase concentration. Using Gibson Assemblage, combine the plasmid, primers, m-cherry, and terminator. Once our product is finished, Use cold, heat shock to insert the plasmid into bacteria. Plate the bacteria on two plates, one containing only LB and the other LB + ampicillin.
June 21st: Yeast Transformation
The plan was to perform yeast transformation on this day but our bacterial transformation was unsuccessful because we had not growth on the plate with ampicillin meaning wither the plasmid did not successfully enter the bacteria or that the plasmid wasn't actually created in the first place.
