Team:WashU/Week9

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Revision as of 20:36, 26 July 2012

Monday, July 23

Today we sonicated three cultures of E. coli - one with our Z construct, one with Z, C and inducers, and one with Z, C, but no inducer. Then, we extracted the soluble proteins from those cultures and ran them on a gel. [PICTURE]

We also ran a PCR of a series of ligations. The color constructs were ligated with promoters into chloramphenicol-resistant plasmid Psb1C3. [EXPAND UPON THIS]

We extracted the proteins from our cultures of E. coli (with our Z construct, with Z and C construct and non-induced, and with Z and C construct plus inducers) and took spectrophotometric readings [SHOW GRAPHS]

Tuesday, July 24

Today, we ordered multiple primers that we will need in the future.

We also performed a ligation of RFP construct with PSL2131, and then we plated the results to check tomorrow.

Wednesday, July 25

We checked the ligation plates of RFP and PSL2131 and see red colonies, indicating that the ligation was successful. After the colonies have a chance to grow a little more, we will start cultures and miniprep them.

We have discovered that we need a fusion protein in order to deal with insoluble protein products that form inclusion bodies, preventing the effective transcription of our gene. Thus, we obtained the pET-42a-c(+) vector, which has a GST tag, from the lab of James Havranek.

We also ran a four-hour experiment to test the effects of our inducers (IPTG and arabinose) on the promoter for the zeaxanthin construct in E. coli. To do this, we varied the concentrations of both of the inducers, with 0 or 1 mM IPTG and 0.02%, 0.2%, and 2% arabinose. We collected OD450 and OD600 measurements every hour. We will take one more reading tomorrow morning and then analyze the data obtained from the experiment. [INSERT THE TABLE AND GRAPHS]

Finally, we digested Psb1C3 with RFP with E and P.

Thursday, July 26

After taking the final OD readings of the E. coli cultures today, we extracted the carotenoids from the tubes and took spectrophotometric readings on them as well.

Start culture for SDS-PAGE gel, once OD600 hits .4, add optimal amount of inducer determined by characterization on Wednesday

Miniprep the ligation of RFP and PSL2131, digest and run on a gel to ensure that the proper ligation occurred

Digestion of Z construct and CS42S + run on gel

Friday, July 27

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 We checked the ligation plates of RFP and PSL2131 and see red colonies, indicating that the ligation was successful. After the colonies have a chance to grow a little more, we will start cultures and miniprep them.
-We have discovered that we need a fusion protein in order to deal with insoluble protein products that form inclusion bodies, preventing the effective transcription of our gene. Thus, we obtained ________ from the lab of James Havranek.
+We have discovered that we need a fusion protein in order to deal with insoluble protein products that form inclusion bodies, preventing the effective transcription of our gene. Thus, we obtained the pET-42a-c(+) vector, which has a GST tag, from the lab of James Havranek.
 We also ran a four-hour experiment to test the effects of our inducers (IPTG and arabinose) on the promoter for the zeaxanthin construct in <i>E. coli</i>. To do this, we varied the concentrations of both of the inducers, with 0 or 1 mM IPTG and 0.02%, 0.2%, and 2% arabinose. We collected OD450 and OD600 measurements every hour. We will take one more reading tomorrow morning and then analyze the data obtained from the experiment. [INSERT THE TABLE AND GRAPHS]