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From 2014.igem.org

We got our sequencing results and aligned them to our plasmid in geneious. 3 out of 4 of the sequencing reactions worked. We plan to sequence the entirety of pHBD1ta or pHBD1tb (transformed in Turbo cells) when the rest of our sequencing primers arrive. We designed primers for hybB cold shock promoter since the iGEM part is not very reliable. We plan to PCR the promoter from the e.coli genome and replace the Tet promoter from pHBD1. We also realized that many of the biobrick parts that we ordered are available in our 2014 kit plate. So we transformed pHBD 9, 10, 12,14, 16 into Turbo cells and plated. We also received our biobrick order from the iGEM headquarters. We plated the stabs for pHBD 19, 11, 4, 18, 13. We also began designing plasmids we want to Gibson in the coming weeks.

Primers for Gibson of hybB promoter in pBbE1a:

HBD15 Fwd on hybB with hom to pBbE1a around pTet TTCTGAAATGAGCTGTCGCCGCTATGGACTGGATAAAG

HBD16 Rev on hybB with RBS and hom to pBbE1a around pTet TTTAAAAGTTTCATATGATATCTCCTGCTACTTAACCCCATGGTGGC

HBD17 Fwd on pBbE1a with RBS and hom to hybB TTAAGTAGCAGGAGATATCATATGAAACTTTTAAAAGTAGCAGCAATTGCAGC

HBD18 Rev on pBbE1a with hom to hybB CAGTCCATAGCGGCGACAGCTCATTTCAGAATATTTGCCAG

Plate in fridge- Turbo, carb, (+), previously arc, pBbE1a-CsgA from 06/09/14 Placed plate in warm room for about 30 minutes Innoculated cells in a solution of 3 mL LB+ 3 microliters of carb Put innoculated cells in tube in warm room and allowed to grow for 2 hours Filled tubes with 15 mL of LB + 15 microliters of carb for paint component to be added Plan: make most concentrated solution of each component and then filter, and then dilute Casein: (50 g/L, 10, 1) Original 500 g/L was too much → made a thick solid in the tube Chitosan: (100 g/L, 10, 1) Gum arabic: (500 g/L, 250, 50) Albumin: (100g/L, 50, 25) Treatments to dissolve the LB+carb+paint component Shaking tubes Water bath at 45 degrees Sonification Autoclave Learned: Chitosan will be very flaky Gum arabic is good at being autoclaved but not at getting filtered Albumin should not be autoclaved Albumin coagulates very easily, when heated Just like frying eggs, which we could have guessed before Casein formed a clump and liquid in the autoclave Allowed turbo cells to grow, and made LB+ carb solution Put cells in cold room to stop growth→ placing in larger flask and growing up more to come tomorrow