From 2010.igem.org
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- Light sensor progress: we had many run-ins with the last ligation
- ‘A&B’ and C were cut both ways:
- ‘A&B’ w/ Eco, Spe and C w/ Eco, Xba
- ‘A&B’ w/ Spe, Pst and C w/ Xba, Pst
- Running diagnostic gel of digest: C w/ EX and AB cut w/ SP worked
- Needed to run it for 2 hrs @ 120V in order to separate the bands effectively because the bp lengths of different parts are too close to each other.
- Extract gel was unsuccessful
- Results: bands were not bright & separate. Top of gel was deformed. Maybe ran too hot for too long? Next time, we will run at 90V or 100V & possibly change out TAE in between.
- The following parts were redigested and ligated: ‘A&B’ w/ ES; C w/ EX and ‘A&B’ SP, C w/ XP, however, they look like they have failed: there were many colonies on the AB (ES) w/ C (EX) plate but more on 3 AB (ES) insert plate. None on AB (ES) insert or AB (SP) vector plate. Very few on AB (SP) w/ C (XP) plate.
- Thus, we propose a new plan of attack:
- We will PCR out inserts, in which we will need to PCR 8 things 4 of ‘A&B’ and 4 of ‘C’
- ON switch progress:
- Interesting stuff: E1010 colonies did not turn red. Will perform 5’ RACE PCR on them
Sent in parts for sequencing and received results for: I746352-B0034, B0034-C0052, C0040-B0015
I746352-B0034 had strange sequence; not even a biobrick was present!
- For now:
- R0065- [B0034-C0052] (SP)-I746352 (XP)-B0034 (ES)-[C0040-B0015] (EX)
B0034 (ES)-C0078 (EX)
- Digested parts: B0034-C0052 (SP), B0034 (ES), C0040-B0015 (EX), C0078 (EX)
- New plan for the off switch looks like this:
- I746351-B0034(ES)-C0078(EX)-R0065-B0034-C0052-I74352-B0034(ES)-[C0040-B0015](EX)
- Made more minipreps of I746352. Will send for sequencing
- Revived parts: I746351 & R0065
- Initiator/Las receiver progress:
- R0082-RBS-RFP was successfully ligated w/ RBS-C0051-B0015.
- PCR screened R0082-RBS-C0051-stop. Miraculously, bands showed up for all 12 samples. And this was done using both the old and new Taq polymerase. Strange. After this, we proceeded to PCR screen R0052-RBS-RFP-RBS-C0051-B0015 and I14015-RBS-C0051-B0015 as well, and we obtained positive results. Time to prepare these for sequencing.
- Next step is to place the I14015-RBS-C0051-B0015 into a carbenicillin backbone because it is currently in a kanamycin resistant backbone. This is necessary because the chassis that we will be transforming these plasmids into are already kanamycin resistant. Thus, we need to replace I14015’s kanamycin resistant backbone.
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We would like to take a moment to thank all of our sponsors for their very generous donations, as we could not have done this without your help!
We would also like to thank and acknowledge:
Our Advisors
Marc Facciotti
Ilias Tagkopoulos
Technical Guidance
David Larsen
Andrew Yao
Visiting iGEMer
Jia Li of Zhejiang University (TEAM ZJU-China)
cI Promoter Screen
Drew Endy - Stanford
Thomas Schneider - NIH
Want to sponsor us? Send an email to mtfacciotti@ucdavis.edu to discuss various ways you can help! :)
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