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Notebook

2022 May

Set up the 2022 SUSTech_Shenzhen iGEM team

Determined that the project is to produce Tyrian Purple in E.coli with a reversible glucose protection

Found PIs to guide and guarantee our TPE project

Applied for funding from School of Life Sciences, SUSTech


2022 Jun

Ordered DNA synthesis during preparation of final exams

Plasmid construction of expressing UGT, PtUGT and BGL

Experimental skills training for beginners


2022 Jul

Obtained FMO, PtUGT and BGL fragments by PCR
Double digested plasmid pET28a
Select the monoclone of transformed E.coli and send for sequencing
FMO and PtUGT were ligated by overlapping PCR
Gibson construction of pET28a-PtUGT-FMO and pET28a-BGL and transformed to E.coli with plate growing
Select the monoclone of transformed E.coli and send for sequencing
Plasmid extraction of pET28a-PtUGT-FMO and pET28a-BGL

2022 Aug

The plasmids Fre-SttH, TnaA-FL-FMO and DH5α strain with endogenous TnaA gene knockout were obtained and transformed into E.coli with plate growing
Select the monoclone of transformed E.coli and cell cryopreserving
Fre-SttH, TnaA-FL-FMO strain plate streaking
Select the monoclone of transformed E.coli and amplification
The bacterial solution is diluted at a fold of 100 and incubated at 37 °C on a shaker at 200 rpm until OD 600 reached 0.6
0.3 mM IPTG is added for induction overnight
Determine soluble and whole cell sample by SDS-PAGE
Fermentation with glucose, Trp and Br-trp at 30 °C
The tyrian purple and indigo product after centrifugation was dissolved by DMSO and purified by washing with a large amount of water
Dye fabric strips with tyrian purple and indigo
SfGFP fragment was obtained by PCR
Double digested plasmid pPOS and pNEG
Gibson construction of pNEG-sfGFP and pPOS-sfGFP
Transform two plasmids in E.coli with plate growing
Select the monoclone of transformed E.coli and send for sequencing
Plasmid extraction of pNEG-sfGFP and pPOS-sfGFP
Prepare pNEG and pPOS competent cells

2022 Sep

Prepare 2xYT medium, pH adjustment and sterilization
pTrpR was transferred into pNEG competent cells
Went through GFP test. Different concentrations of IPTG were added for induction with adding the substrate Br-Trp, and the fluorescence intensity was detected by microplate reader
The emulsion was configured and mixed with the cell suspension induced to produce sfGFP, and the quality of emulsion was detected by phase contrast microscopy
INAC-pTrpR was constructed by inserting random sequences into pTrpR
INAC-pTrpR and pTrpR were mixed into 1:1, 1:10, 1:100 and 1:1000, and SDS-PAGE was performed after PCR
INAC-pTrpR and pTrpR were mixed at the ratio of 1:1 and converted into pNEG competent cell.
CPR cycle was performed, and the product was collected to run agarose electrophoresis
The colonies on the plate were scraped to buid up formal library
Plasmid extraction to obtain No.0 library
No.0 library was transformed into competent cell transformed with pNEG
CPR cycle was performed, and product was cleaned and concentrated using a PCR purification column after run a agarose electrophoresis
Assemble DNA product in 0928 with pTrpR backbone to get No.1 library.

Future Plan

Check the size and analyze the selection result of No.1 library

Transform pET28a-PtUGT-FMO and pET28a-BGL into BL21 competent cell and try to produce indican

Supplement quantitative experiments to fermentation of producing tyrian purple by LC-MS
Continue directed selection of No.1 library, and check the size and analyze the selection result of library in each cycle

Find other residue combinations, and construct new library for selection

Use the muted trp repressor to construct sensor system to test promotor expression in different ratio of trp and br-trp