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Team:Prairie View/Notebook
From 2008.igem.org
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Notebook
Molecular Biosensor
July
PlanningWe began our project by discussing the function and interaction of an Electronic Nose (E-Nose) for assessing the parameters of data results. ENOSE FUNCTION Through planning out our laboratory project, we decided that designing our parts in a way that they can be assembled in a modular fashion could best be achieved by using compatible restriction enzymes site to flank each gene. We then had to research the process of PCR to get a better understanding of how to design primers for this approach, compiled laboratory techniques that we would be using during the project, and consulted the literature for ideas on design and assembly.
August
Primer Design, PCR and DigestionWe designed primers for each part that was synthesized for amplification in PCR. An oligo was designed containing the RBS sequence and was surrounded with compatible restriction sites which would be ligated in front of each part. After running PCR with our designed primers on each part, we digested them to be ligated behind an RBS and into our expression vector. Performing this two step ligation connect each part to a RBS proved complicating. Through multiple attempts, we tried varying the concentrations and volumes for the ligation but received few results.
Figure 1 PCR 9/25/08
Marker, Cytochrome, Vanadium, Calcium, FhuA, Ton B, Nickel B, Nickel A
Figure 2 Parts1 (9.25.08) This gel shows our PCR digested. 8. Cytochrome C: 354 7. Calcium: 756 6. Ton B: 768 5. Vanadium: 1254 4. Nickel A: 1614 3. Nickel B: 978 2. FhuA: 2277 1. Marker
Figure 3 DigPCRparts(9.30.08)
Figure 4DigPCRparts(9.30.08)
Only Nickle A, Cytochrome C, and Ton B, And FhuA were the only parts that showed consistenly. Cytochrome C and Ton B showed but at the wrong sizes.
September
Redesigning of primers and Assembly/Digesting and Ligation.Start: To eliminate complications of ligating parts to a RBS, we designed new primers that included the RBS sequence into the PCR product. By doing this, we avoided the two-step ligation process. Some of the genes that we were going to include into our ligations contained the same restriction enzyme sites internally that are used to assemble the parts together. To avoid digesting our gene in the assembly process, one point mutations had to be introduced that would change one base of the restriction site while maintaining the same translated amino acid. To do this, mutation primers were designed and run on PCR to introduce the point mutations.
Figure 10 PCR Mutation (10.1.08)
Finish:
Figure 8 DigPlasmids2(10.22.08)
Figure 93DigRep (10.7.08)
Figure 10 PartsBgl2-EcoRI(10.1.08)
October
- Testing Ligations and inputting data for E-Nose AnalysisWe tested our ligations in vivo. The testing was composed of growing up ligation expressing cultures, taking samples at 0,12,18,24,36, and 48-hours and extracting their plasmids. Measurements were taken of OD, DNA concentration, DNA fluorescence and ATP concentration to determine the effects of the ligations within the cell. DATA
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