Team:NCKU Tainan/Notebook Construction

Notebook Construction - iGEM NCKU

NOTE / Construction
Notebook - Construction
Extracted plasmid from the bacteria provided by XMU
(pSB1C3-BBa_B0034, pSB1C3-BBa_B0015, pSB1C3-BBa_K592009)
2016.03.30

We got the bacteria with Biobrick from XMU.

2016.04.01

Pre-culture: 3 ml of LB (with Chloramphenicol) was inoculated with one colony from LB agar plate.

2016.04.02

Culture: 10 ml of LB (with Chloramphenicol) culture with 100 ml/μL pre-cultured bacteria.

And grow up at 37 degree for 10-12 hours.

2016.04.03

Extracted the plasmid, and measured the concentration.

Store the plasmid at -20 degree.

Got the glucose sensitive promoter (PpI, Pcar) by PCR
2016.04.16

PCR for PpI & Pcar.
Result: After gel purified, the concentration of the DNA was too low to do construction.

2016.04.17

PCR for PpI & Pcar again (in big scale).

Purified the PCR product and cloned to T-vector.

2016.04.18

Confirmed the white colony by PCR (using M13F & M13R as the primer)
Result: Some of the colonies were correct.

Pre-culture: 3 ml of LB (with Ampicillin) was inoculated with one correct colony from the agar plate.

2016.04.19

Extracted plasmid and confirmed plasmid size by gel electrophoresis.
Result: The size of pMD19T-PpI and pMD19T-Pcar were correct.

2016.04.20

Culture: 10 ml of LB (with Ampicillin) cultured with 100 ml/μL pre-cultured bacteria.

Grow up at 37 degree for 8 hours and stored the bacteria at -70 refrigerator.

Constructed pSB1C3-B0034-K592009
2016.04.30

Digested pSB1C3-B0034 (SpeI,PstI) and pSB1C3-K592009 (XbaI,PstI)

Ligated K592009 (insert) to pSB1C3-B0034 (vector) and transformed the product.

2016.05.01

Confirmed by colony PCR (using Prefix-F & Suffix-R as the primer).

Pre-culture: 3 ml of LB (with Chloramphenicol) was inoculated with one correct colony from the agar plate.

2016.05.02

Extracted plasmid and confirmed plasmid size by gel electrophoresis.
Result: The size of pSB1C3-B0034-K592009 was correct.

2016.05.03

Culture: 10 ml of LB (with Chloramphenicol) cultured with 100ml/μL pre-cultured bacteria.

Grow up at 37 degree for 8 hours and stored the bacteria at -70 refrigerator.

Construct pMD19T-Pcar-B0034-K592009 and pMD19T-PpI-B0034-K592009
2016.05.07

Digested pSB1C3-B0034-K592009 (XbaI,PstI), pMD19T-Pcar (SpeI,PstI) and pMD19T-PpI (SpeI,PstI)

Ligated B0034-K592009 (insert) to pMD19T-Pcar (vector) and pMD19T-Pcar (vector).

Transformed the product.

2016.05.08

Confirmed by colony PCR (using M13F & M13R as the primer).

Pre-culture: 3 ml of LB (with Ampicillin) was inoculated with one correct colony from the agar plate.

2016.05.09

Extracted plasmid and confirm plasmid size by gel electrophoresis.
Result: The size of pMD19T-Pcar-B0034-K592009 and pMD19T-PpI-B0034-K592009 were correct.

2016.05.10

Culture: 10 ml of LB (with Ampicillin) cultured with 100 ml/μL pre-cultured bacteria.

Grow up at 37 degree for 8 hours and stored the bacteria at -70 refrigerator.

We transformed BioBricks from the 2016 igem distribution
(BBa_J23110, BBa_J23101,BBa_B0031,BBa_B0032,BBa_E1010)
2016.06.03

Transformed 3 ml/μL biobrick to 100 ml/μL DH5α competent cell by heat shock.
Result: The transformation of BBa_J23110, BBa_B0032 and BBa_E1010 was successful.
Result: The transformation of BBa_J23110, BBa_B0032 and BBa_E1010 was fail.

2016.06.04

Pre-culture: 4 ml of LB (with Chloramphenicol) was inoculated with one colony from LB agar plate.

Extracted plasmid by Plasmid miniPREP Kit and confirmed plasmid size by gel electrophoresis.

2016.06.05

Culture: 10 ml of LB (with Chloramphenicol) was inoculated with one colony from LB agar plate. And grow up at 37 degree for 10-12 hours.

2016.06.06

Extracted the plasmid, and measure the concentration.

Stored the plasmid at -20 degree.

2016.06.07

Transformed 5 ml/μL biobrick to 100 ml/μL DH5α competent cell by heat shock.
(BBa_J23110, BBa_B0032 and BBa_E1010)
Result: The transformation of BBa_J23110, BBa_B0032 and BBa_E1010 were successful.

2016.06.08

Pre-culture: 4 ml of LB (with Chloramphenicol) was inoculated with one colony from LB agar plate.

Extracted plasmid by Plasmid miniPREP Kit and confirmed plasmid size by gel electrophoresis.

2016.06.09

Culture: 10 ml of LB (with Chloramphenicol) was inoculated with one colony from LB agar plate.

And grow up at 37 degree for 10-12 hours.

2016.06.10

Extracted the plasmid, and measure the concentration.

Stored the plasmid at -20 degree.