Team:CSU Fort Collins/Notebook/KillSwitch/Jul

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<h4>Tuesday, July 29</h4>
<h4>Tuesday, July 29</h4>
Prepared overnight culture with LB medium, CM and colonies 11 and 13.
Prepared overnight culture with LB medium, CM and colonies 11 and 13.
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<h2>AUGUST</h2>
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<h4>Friday, August 1</h4>
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Enzyme digestion of puc57-KillerRed linearized plasmid backbone using EcoR1 and Pst1 and PSB1C3 using Xba1 and Spe1
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 +
<h4>Thursday, August 7</h4>
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Ligated KillerRed into PSB1C3
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<h4>Tuesday, August 12</h4>
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Transformed previous ligation assuming KillerRed would not function without a promoter. Did not successfully yield colonies.
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 +
<h4>Monday, August 25</h4>
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Ligated two samples of KillerRed with Puc57.
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We decided to test whether the KillerRed ligated into the backbone it came with would be transformable.
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<h4>Tuesday, August 26</h4>
 +
Transformation of linearized plasmid backbone and puc57 KillerRed. Did not yield colonies.
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 +
 +
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Revision as of 20:06, 17 October 2014

Kill Switch Notes - July

Kill Switch Daily Notes

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July

TRP/KillerRed Assembly

August

The Month of Failed Transformations

September

Troubleshooting and Biobrick Success

JULY

Tuesday, July 1 - Monday, July 7

Contacted Dr. Peebles about possible lead. When E. Coli containing the light sensitive gene visA is exposed to intense white light it overproduces protoporphyrin which then inhibits the correct assembly of the cell membrane. This overproduction causes a buildup of oxygen in the cell causing the cell to rupture. There is an iGEM Bio-Brick attached to this gene (also called the HemH gene) here.

Tuesday, July 8

We looked through registry of standard biological parts from iGEM and we found four possible promoters. We assessed these based on sample availability, uses, experience logs as well as their tendency for leakage.

Wednesday, July 9

Upon recommendation of Dr. Peebles, decided to pursue creation of a KillerRed protein Bio-Brick. The tryptophan (TRP) promoter maybe a good option to control KillerRed. In the presence of tryptophan, a repressor binds to the promoter and inhibits gene expression. When tryptophan is absent, the repressor cannot bind to the promoter and this allows gene expression.

Tuesday, July 15

Miniprep on Trp and GFP. Measured concentrations via nanodrop.

Thursday, July 17

Completed an Enzyme Digestion on Trp and GFP.

Friday, July 18

Ligated Trp + GFP via the Cloning a Gene into a Plasmid protocol. Incubated over the weekend.

Tuesday, July 22

Transformation of Trp + GFP into Puc 19 and plating onto chloramphenicol (Cm) resistance.

Wednesday, July 23

Conformation of construction and colony PCR on successful plates.

Thursday, July 24

Ran successful gels on colonies.

Tuesday, July 29

Prepared overnight culture with LB medium, CM and colonies 11 and 13.

AUGUST

Friday, August 1

Enzyme digestion of puc57-KillerRed linearized plasmid backbone using EcoR1 and Pst1 and PSB1C3 using Xba1 and Spe1

Thursday, August 7

Ligated KillerRed into PSB1C3

Tuesday, August 12

Transformed previous ligation assuming KillerRed would not function without a promoter. Did not successfully yield colonies.

Monday, August 25

Ligated two samples of KillerRed with Puc57. We decided to test whether the KillerRed ligated into the backbone it came with would be transformable.

Tuesday, August 26

Transformation of linearized plasmid backbone and puc57 KillerRed. Did not yield colonies.



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