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From 2014.igem.org

Finished the minipreps of BBa_K274200, BBa_I765001, pHBD4, BBa_K1073023, BBa_K1033931, and BBa_K1033929.

Plated Gibson transformations of pHBD24 and pHBD21 had few (1-2) colonies. We ran diagnostic colony PCRs on one colony on each plate to check for the inserts

We then ran them on a gel. Although bands did show up, they were not of the size we were expecting (we should have seen bands at about 3 kb for both plasmids). Although confused as to why there were bands, we don’t believe our Gibsons worked.

We decided to run the Gibsons for pHBD24 and 21 again as well→ transformed→ plated

We ran the gradient PCR for the HSP and ASP reactions on a gel. The image showed that the HSP and ASP inserts amplified however the backbone failed to amplify as well as the Blux insert.

We decided that we needed to design new primers for the backbone and insert.

We plated the new chromoprotein biobricks (eforred, aeblue, yellow(GFP)) that were picked up from the igem hq as well as pHBD4 that was retransformed. We ran the PCR for pHBD39-41(pBbE1a_Tet_Chromoprotein) Gibsons (See PCR planning spreadsheet for details) to get fragments and backbones with gibson tails.

We also set up another miniprep on BBa_K1073023 and BBa_K1033929 because their earlier minipreps were contaminated.

Activity Log Checked on the results of the biocompatibiltiy assay Only plates where E. coli grew were the coconut oil and gum arabic plates We think this might be due to the presence of tween 20 Made new solutions to test Control: LB + 100mg/mL carbenicillin Kaolin powder (additive) 100g/L of powder, LB + 100mg/mL carbenicillin Coconut oil + gum arabic 15% coconut oil, 3g/20mL gum arabic, LB + 100mg/mL carbinicillin Coconut oil + gum arabic + Kaolin 30 mL LB→ with 100mg/mL of carbinicillin 15% coconut oil (4.5 mL) 4.5 g gum arabic 3 g kaolin Finding binding domains to order-> Goal to have them ordered for tomorrow→ start working with them on Monday