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From 2014.igem.org

Our LacO hypothesis was rejected as none of the IPTG induced cultures showed any coloring.

However, we noticed that one of our plates with pHBD44 had blue colonies! Even though it was in the 4C refrigerator, the colonies became blue (just took overnight, about 24 hours from plating).

We finished minipreps of pHBD 37 and 44.

Began the PCR to create Gibson fragments for pHBD 43 (PCRed pHBD 37 and 1 with appropriate primers).

Gel for biobrick cloning reaction was left overnight...saw some desired bands except for ASP. The PCRs for the Gibsons of pHBD 26 and 28 didn’t work because only the pHBD 18 fragment showed up (lane 12):

Decided to rerun the digest:

Gel extracted visible correct bands for HSP, RFP, and backbone. However, the concentrations were too low. Figured out that it was because we were digesting 250ng of DNA instead of 1-2ug needed for a gel extraction. To do a 1-2ug digestion, we need more pHBD35 so we decided to retransform from our stocks and grow up overnight (tomorrow night).

We also decided to redo the PCRs for the Gibsons of pHBD 26 and 28 (pHBD 18, 13, 35) because they didn’t work.

We then ran the Gibson fragments of pHBD 37 on a gel:

Since these were successful, we gel extracted the fragments, then Gibsonned them to create pHBD 43. We then transformed pHBD 43 and plated it.

We also retransformed plasmids we wanted to make glycerol stocks for: pHBD1, 35, 44, and 2.

We have a new hypothesis for why our pBbE1a_Tet_Chromoprotein constructs sequenced correctly but are not producing color. The prefix that we use as our gibson tail is probably adding too much distance between the RBS and start codon. We designed primers to clone out the prefix in some of the chromoproteins:

HBD65 AAGAGGAGAAATACG ATGAGTGTGATCGCT 60.5 Fwd prefix removal pHBD39 CPBlue HBD66 AGCGATCACACTCAT CGTATTTCTCCTCTT 60.5 Rev prefix removal pHBD39 CPBlue HBD67 AAGAGGAGAAATACG ATGGCTTCCTCCGAA 62.1 Fwd prefix removal pHBD40 HBD68 TTCGGAGGAAGCCAT CGTATTTCTCCTCTT 62.1 Rev prefix removal pHBD40 HBD69 AAGAGGAGAAATACG ATGGCTTCCAAAATA 58 Fwd prefix removal pHBD41 HBD70 TATTTTGGAAGCCAT CGTATTTCTCCTCTT 58 Rev prefix removal pHBD41