Harvard BioDesign/8 July 2014

From 2014.igem.org

Gel extracted parts for HybB + SB3K3 for Gibson. Ran Gibson. Transformed.

Picked colonies from PCR of pHBD39-41 to clone out prefix. Only pHBD41 had many colonies. Picked 2 colonies from pHBD41 and one colony from pHBD40 to grow up for miniprep. (We forgot to do this miniprep that day).

Heat Shock Promoter Assay:

Put 20uL of pHBD26 which was grown up overnight in 10mL media into 1mL of LB

Approach 1:

In 3 replicates:

Incubate shaking for 1, 2, 3, 4, 5, & 6 hours at 25C-37C-42C Inoculate 3 samples with just cells right before read Take 100uL of each into well of 96-well plate and take measurements in plate reader

Approach 2:

In duplicates:

Heat shock for 10 minutes at 25C-37C-42C Take 100uL into well of 96 well plate and take measurements in plate reader every 15 minutes for 6 hours.

(Insert Data here)

Transformed pHBD41 and 43 (blue and yellow chromoprotein) into LSR10 cells.

15:20, 17 October 2014 (CDT)15:20, 17 October 2014 (CDT)15:20, 17 October 2014 (CDT)15:20, 17 October 2014 (CDT)15:20, 17 October 2014 (CDT)15:20, 17 October 2014 (CDT)15:20, 17 October 2014 (CDT)15:20, 17 October 2014 (CDT)15:20, 17 October 2014 (CDT)Mmmlong

Plated LSR10 and Turbo cells from biocompatibility assay Plated LSR10, with autoclaved paint components (2) Plated LSR10, with non-autoclaved paint components (1) Plated Turbo with autoclaved paint components

Performed Gibson reaction Used gblock inserts and linearized F12 backbone 4 microliter of insert to Gibson (0.1 pmol) 4 microliters of backbone to Gibson (0.03 pmol) 5 microliters of water 10 microliters master mix

GBLOCK CBM_1_1 CBM1_2 ChiA1 ChiC_BD Length 162 162 195 183 Amount (ng) 200 200 200 200 Molecular Weight 99,993.4 99,997.3 120,389.5 112,966.8 Concentration (fmoles/ng) 10 10 8.31 8.85

Ran gel on plasmids resulting from Gibson Bands were a little over 4,000 base pairs Backbone is around 4,000 bp Inserts were 35-45 bp long Gel extracted plasmids