"

From 2014.igem.org

Promoter Cloning (HSP & ASP into SB3K3 backbone):

We looked at our cultures that we placed in the 25 C and 30 C shakers over the weekend, but there was no color. We weren’t sure why, but we hypothesized that there might still be a LacO site that could be inhibiting the promoters.

We picked colonies from pHBD39-pHBD42 into 5 ml of culture. We grew them up for 4 hours at 37 C and then induced with 0.25 mM of iPTG and moved to the 25 C shaker for overnight. We hoped that inducing with iPTG would get rid of any inhibitory effect that the LacO site was having.

Set up PCR to create the Gibson fragments for pHBD 26 and pHBD 28. So, PCRed pHBD 18, 13, and 35 with appropriate primers. Left PCR at 4C overnight.

Tried biobrick cloning method to get HSP and ASP + RFP into SB3K3 backbone. Digested promoters with EcoRI and SpeI, RFP with XbaI and PstI, backbone with EcoRI and PstI. See spreadsheet for specific reactions. Ran on a gel (to be imaged tomorrow).

We began the overnight cultures for miniprep of pHBD 37 and pHBD 44.

Paint: Designed and ordered 4 gBlocks: 2 containing cellulose-binding domains, and 2 containing chitin-binding domains. gBlocks were ordered with end homologies to a 12-amino acid linker sequence present on a variant of our pBbe1A plasmid. We also designed primers to be used in a Gibson assembly to switch out the 12-aa linker for a 48-aa linker, to allow us to determine the optimal linker length. Since sequencing revealed mutations in the plasmid with a 24-aa linker, we do not plan to initially test the effectiveness of a 24-aa linker. If the 12 and 48 amino acid linkers result in proteins with significant functional differences and neither is optimal, we may investigate the use of a 24-aa linker.

Gibson/PCR primers (same reverse primer used for all reactions):

CBM1-1 F48 primers

F GGTAGCGGCGGTGGATCTGGCGGCGGCTCTACCCAGTGGGGCCAGTGCGG R TTTGATGCCTGGAGATCCTTACTCGAGTCA

CBM1-2 F48 Primers F GGTAGCGGCGGTGGATCTGGCGGCGGCTCTACCCATTATGGCCATCGCGG

ChiA1 F48 F GGTAGCGGCGGTGGATCTGGCGGCGGCTCTGCGTGGCAGGTGAACACCGC

ChiC_BD F48 F GGTAGCGGCGGTGGATCTGGCGGCGGCTCTCCGGAATGGGATCCGGATAC