Team:HKUST/Notebook

14/06/2010
BioBrick BBa_I746101 was extracted from 96 wells plates. E.coli DH10B competent cells were successfully transformed with BioBrick BBa_I746101 which contains agrC.

25/06/2010
PMH4 containing mCherry and PBI121 conatining gusA reporter gene were successfully transferred into E.coli DH10B competent cells.

12/07/2010
Fusion PCR of agrC-mCherry was performed and products were confirmed by agarose gel electrophoresis.

30/07/2010
Genomic DNA of Lactobacillus. Plantarum WCFS1was extracted. Next, PCR of plnB from the genomic DNA was conducted and products were confirmed by agarose gel electrophoresis.

21/07/2010
Successfully extract plasmid pMG36e out from filter paper.

22/07/2010
E.coli DH10B competent cells were successfully transformed with pMG36e

28/07/2010
Enzyme digested the vector and confirmed plasmid pMG36e.

10/08/2010
Midi-prep of shuttle vector pMG36e was performed and extracted plasmids were confirmed by enzyme digestion test.

16/08/2010
PCR of gusA from PBI121 was conducted and products were confirmed by agarose gel electrophoresis.

17/08/2010
Fusion PCR of agrC-plnB was performed and products were confirmed by agarose gel electrophoresis.

28/08/2010
Primer (*****-*****) arrived and repare primers for ligation. Double strand DNA of FLAG-tag, both part 1&2 of signal peptide and DD13-RIP was obtained.

20/08/2010
Double-strand DNA of plnA promoter (124bp) was obtained. By following the protocol of fusion PCR, annealing of the forward primer (79bp) with the reverse primer (73bp) and the extension were accomplished.

26/08/2010
agrC-plnB was ligated into pBluescript KS (+). Ligation products were transferred into E.coli DH10B competent cells by heat shock transformation. Ligation was confirmed by colony PCR and plasmid digestion test.

13/09/2010
agrC was ligated into pBluescript KS (+). Ligation products were transferred into E.coli DH10B competent cells by heat shock transformation. Ligation was confirmed by colony PCR and plasmid digestion test.

20/09/2010
Performed three-way ligation of SP with plasmid pMG36e.

22/09/2010
Colony PCR shown that the insert was successfully ligated into plasmid pMG36e.

26/09/2010
Sequencing of the insert was obtained by DNA sequencing to further confirm the ligation product.

24/09/2010
gusA was ligated into backbone pSB1C3. Ligation products were transferred into E.coli DH10B competent cells by heat shock transformation. Ligation was confirmed by colony PCR and plasmid digestion test.

29/09/2010
Two constructs agrC and agrC-plnB were transferred from pBluescript KS (+) to shuttle vector pMG36e. The ligation products were confirmed by colony PCR and enzyme digestion.

05/10/2010
Sequencing of four constructs: agrC in pBluescript KS (+), agrC-plnB in pBluescript KS (+), agrC in pMG36e and agrC-plnB in pMG36e. All of them were finally confirmed.

11/10/2010
mCherry was ligated into pBluescript SK (+). Some colonies on the plate showed red color expressed by mCherry.

12/10/2010
GUS assay with the substrate X-Gluc. E.coli containing PBI121 gave more gusA expression than control group.
agrC-plnB (for localization test) was ligated into pBluescript SK (+). Ligation product was confirmed by colony PCR and enzyme digestion.

13/10/2010
GUS assay with the substrate 4-NPG. E.coli containing PBI121 gave more gusA expression than control group.

15/10/2010
Construct agrC-plnB-mCherry was built by ligating mCherry into pBluescript SK (+) containing agrC-plnB already.