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Team:WarrenCIndpls IN-HS/Notebook
From 2011.igem.org
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Contents |
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. Once the DNA separated, we froze it until the following Monday.
