iGEM 2010 — Team HKUST

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# Team:HKUST/Notebook

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Team: HKUST

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* [HOME](https://2010.igem.org/Team%3AHKUST)
* [Here we are!](https://2010.igem.org/Team%3AHKUST/Team)
* [Accomplishment](https://2010.igem.org/Team%3AHKUST/Accomplishment)
* Project* * [Abstract](https://2010.igem.org/Team%3AHKUST/Project)
    * [Background](https://2010.igem.org/Team%3AHKUST/Project/Background)
    * [Experimental Design](https://2010.igem.org/Team%3AHKUST/Project/Experiment%20Design)
    * [Experimental Results](https://2010.igem.org/Team%3AHKUST/Project/Results%20and%20Discussion)
    * [Materials and Methods](https://2010.igem.org/Team%3AHKUST/Project/Materials%20and%20Methods)
    * [Future Work](https://2010.igem.org/Team%3AHKUST/future_work)
    * [BioBricks](https://2010.igem.org/Team%3AHKUST/biobrick)
    * [Human Pratice](https://2010.igem.org/Team%3AHKUST/Human_Practice)
    * [Lab Notebook](https://2010.igem.org/Team%3AHKUST/Notebook)
    * [Contributions](https://2010.igem.org/Team%3AHKUST/attribution)
    * [Acknowledgement](https://2010.igem.org/Team%3AHKUST/Acknowledgement)
    * [Biosafety](https://2010.igem.org/Team%3AHKUST/Safety)

# Lab 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

Plasmid PMH4 containing mCherry and plasmid 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.

21/07/2010

Successfully extract plasmid pMG36e out from filter paper.

22/07/2010

Transform  *E.coli* DH10B competent with plasmid pMG36e.

28/07/2010

Plasmid pMG36e was enzyme digested and confirmed..

30/07/2010

Genomic DNA of *Lactobacillus Plantarum* WCFS1 was extracted. Next, PCR of *plnB* from the genomic DNA was conducted and products were confirmed by agarose gel electrophoresis.

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.

20/08/2010

Double-stranded DNA of *plnA* promoter (124bp) was obtained.

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.

28/08/2010

Primers arrived and prepareed primers for ligation. Double strand DNA of FLAG-tag, both part 1&2 of signal peptide and DD13-RIP was obtained.

12/09/2010

*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.

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.

26/09/2010

Sequencing of the insert (*gusA* inserted into pSB1C3) was obtained by DNA sequencing to further confirm the ligation product.

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 was performed. *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.