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Team:WarrenCIndpls IN-HS/Project
From 2011.igem.org
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Some bacteria have been genetically modified to detect certain metals in a water supply. However, it has not been done in yeast. If yeast is genetically engineered to do so, it would be a much safer procedure. The objective for was to create a bio-brick. The creation of the part will enable next year's team to complete the overall goal, which is to create and insert a plasmid into yeast that will cause it to glow red in the presence of a certain metal. | File:WarrenCIndpls IN-HS team.png Your team picture |
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Contents |
Overall project
Project Details
Gibson Assembly
Gibson Assembly, a relatively new method of combining DNA, was used
The Experiment
DNA Extraction:
1) Pipet 200 microliters of distilled water into two centrifuge tubes
2) Add a drop of detergent to each tube and shake.
3) Label the tubes according to what will go into each.
4) Rub the loop end of the inoculating loop across the surface of the agar with the E.Coli containing the ADH terminator.
5) Place the loop into the corresponding tube and swivel the loop a few times.
6) Repeat 4-5 with the E.Coli containing M-Cherry.
7) Shake the tubes well or place them on a vortex.
8) Place in a hot water bath at 56-60 degrees Celsius for 10 minutes
9) Add a pinch of meat tenderizer to each tube. -this will break apart the proteins holding the cells together.
10) Vortex the solutions again.
11) Place them in a water bath at 56-60 degrees Celsius for 20 minutes
12) Pipet 200 microliters of ethyl alcohol(cold) into each tube and allow separation to take place for 15 minutes.
DNA Amplification (PCR)
1) In each tube, there will be three layers of materials; using the pipet, collect the first and half of the second layers of each tube.
2) Place the collected material into a different centrifuge tube each. (Label)
3) Spin both tubes down using the centrifuge.
4) Pour off the supernatant.
5) Resuspend the DNA pellets in each tube by adding 200 microliters of distilled water and vortexing.
6) Using the same method as before, extract the plasmid(Prs 416) from bacteria.
7) Pipet 2 microliters of forward primer of RFP, 2 microliters of reverse primer of RFP, 2 microliters of reverse primer of ADH-1 Terminator, 5 microliters dNTP, 1.5mM Magnesium Chloride, 10 microliters of 10X Buffer, 1 microliter of DNA, 1 microliter of Taq, and 73 microliters of distilled water.
8) Run a PCR; 25 cycles, with denature at 94 degrees Celsius for 40 seconds, anneal at 58 degrees Celsius for 1 minute, and extension at 68 degrees Celsius for 2 minutes, then 68 degrees for 15 minutes.
Gibson Assembly
1) While the PCR is running, prepare the master mix for Gibson.
2) Pipet 80 microliters of 5X ISO Buffer, 0.16 microliters of T5 exonuclease, 5 microliters of Phusion Polymerase, 40 microliters of Taq ligase, and 174.84 microliters of distilled water. Freeze until needed later.
3) Also, using the same method as before, extract the plasmid(Prs 416) from bacteria.
4) Once PCR is done, run the Gibson using the PCR machine.
5)
Results
In order to transform into yeast and complete our objective, we first had to transform into bacteria. Because the bacteria didn't grow until a few days after being plated, the results were inconclusive and we couldn't continue on.
