Team:TP CC SanDiego/Notebook
NOTEBOOK
DAY 1
SUMMARY (July 15th):
PCR Amplified pPBR322-based plasmid backbone, pBAD from E. coli gDNA, and rrnB from the F plasmid.
DPN1 digest for 1 hour at 37°C and PCR Purification using Qiagen Spin Columns.
Gel analysis for correct PCR amplification of fragments
WHAT TO DO NEXT:
Gibson Assembly all fragments into complete plasmid for diagnostics.
DAY 2
SUMMARY (July 18th):
Gibson Assembly of vector backbone, pBAD, rrnB, and placeholder YFP (in between pBAD and rrnB) gene fragments (30 fmol each).
Electroporated assemblies into electrocompetent Epi300 cells; recovered for 1 hour at 37°C and plate on LB+Tet plates.
WHAT TO DO NEXT:
Colony PCR of colonies on plate.
DAY 3
WHAT WE FOUND:
Numerous colonies (~40) to screen.
SUMMARY (July 19th):
Colony PCR of 16 colonies using screening primers to test accurate assembly across pBAD-YFP-rrnB junctions.
WHAT TO DO NEXT:
Restart positive colonies for miniprep.
DAY 4
SUMMARY (July 20th):
Restarted four positive colonies in 5 mL of LB+Tet in 37°C for minipreps tomorrow by sampling from patches made during colony PCR.
WHAT TO DO NEXT:
Plasmid miniprep from restarted cultures.
DAY 5
SUMMARY (JULY 21st):
Extracted pBAD plasmids from cultures using Qiagen Miniprep kit; plasmid extracts were nanodropped to find plasmid concentrations.
Plasmids were HindIII digested to ensure final proper plasmid construction.
WHAT TO DO NEXT:
Amplify vector backbone for chitinase plasmids; replace YFP with chitinase inserts.
DAY 6
SUMMARY (AUGUST 12th):
Designed inserts were ordered from IDT (LbCHI31 and LbCHI32)
red pcr tube in -20C on igem rack contains product
WHAT TO DO NEXT:
Transformation into BL21 and Dh5Alpha competent cells
Make gels
DAY 7
SUMMARY (August 15th)
Inserts arrived from IDT; resuspended in H2O.
Gibson Assembly of LbCHI31 and LbCHI32 fragments into pBAD vector backbone.
Electroporated assemblies in electrocompetent Epi300 cells and recovered for 1 hour at 37°C before plating on LB+Tet plates.
WHAT TO DO NEXT:
Colony PCR of LbCHI31 and LbCHI32 plasmid transformed colonies.
DAY 8
WHAT WE FOUND:
6 LbCHI31 and 17 LbCHI32 colonies to screen.
SUMMARY (August 16th)
Colony PCR of 16 colonies each for LbCHI31 and LbCHI32 plates using screening primers to test accurate assembly across pBAD-chitinase-rrnB junctions.
11 of LbCHI31 and 15 of LbCHI32 colonies were found to be positive.
WHAT TO DO NEXT:
Restart positive colonies for miniprep.
DAY 9
SUMMARY (August 17th)
Restarted four positive colonies each from LbCHI31 and LbCHI32 patches in 5 mL of LB+Tet in 37°C for minipreps tomorrow by sampling from patches made during colony PCR.
WHAT TO DO NEXT:
Plasmid miniprep from restarted cultures.
DAY 10
SUMMARY (August 18th)
Extracted LbCHI31 and LbCHI32 plasmids from cultures using Qiagen Miniprep kit; plasmid extracts were nanodropped to find plasmid concentrations.
Plasmids were HindIII digested to ensure final proper plasmid construction. All 8 digests turned up positive.
WHAT TO DO NEXT:
Transform plasmids into E. coli BL21 for protein expression analysis.
DAY 11
SUMMARY (August 23rd)
LbCHI31 and LbCHI32 plasmids (two positive colonies each) were transformed via heat shock into chemicompetent BL21 cells.
Cells were recovered for 1 hr at 37°C and plated on LB+Tet to be incubated at 37°C overnight (total 4 plates).
WHAT TO DO NEXT:
Prepare glycerol stocks of colonies that grow on the plates.
DAY 12
SUMMARY (August 24th)
Glycerol stocks were prepared for 16 colonies (4 per plate, 4 plates with 2 each for LbCHI31 and LbCH32 BL21 samples).
WHAT TO DO NEXT:
Restart glycerol stocks at a later date for protein expression.
DAY 13
SUMMARY (October 12th)
Glycerol stocks for 2 LbCHI31 and 2 LbCHI32 colonies were restarted in 10 mL LB+Tet at 37°C overnight.
WHAT TO DO NEXT:
Induce cultures with 1% L-arabinose to promote chitinase production and secretion.
DAY 14
SUMMARY (October 13th)
Cultures were split in half (two samples per culture of 5 mL each); raised the volume of each split culture to 10 mL with Lb+Tet and recovered for 1 hr at 30°C prior to induction.
One copy of each culture set was induced with 1% L-arabinose overnight at 30°C to avoid protein misfolding and aggregation. Other uninduced cells were incubated at 30°C overnight as well.
WHAT TO DO NEXT:
Prepare cells for protein expression analysis.
DAY 15
SUMMARY (October 14th)
All induced and uninduced cultures were spun down, and supernatants were aspirated and preserved separately for further tests to gauge secretion of the chitinase proteins. The pellets were frozen at 80°C for preservation until SDS Page preparation.
WHAT TO DO NEXT:
Prepare samples for SDS Page and protein expression analysis.
