Team:NCKU Tainan/Notebook

Notebook

Construction of BBa_K2275007

5/22

  • PCR to get DNA of nir gene cluster from Eschericha coli MG1655 and analyze by DNA gel.

5/23

  • Digest nir gene cluster and pSB1C3-PLacI-sfGFP with HindⅢ and SpeI. Analyze by the DNA gel.
  • Ligase at 16℃ overnight.

5/25

  • Transform the ligased product into DH5α and put at 37℃ for 12 hours.

5/26

  • Select five colonies from the plates and pre-culturing.

5/27

  • Extract plasmid and use plasmid digestion to confirm.

Construction of BBa_K2275008

7/3

  • PCR to get DNA of gudB from Bacillus subtilis and analyze by the DNA gel.
  • Digest gene gudB and pSB1C3-PLacI-sfGFP with BamHI and PstI. Analyze by the DNA gel
  • Ligase at 16℃ overnight.

7/4

  • Transform the ligased product into DH5α and put at 37℃ for 12 hours.

7/5

  • Confirmation by colony PCR.
  • Select two colonies from the plates and pre-culturing.

7/6

  • Extract plasmid and use plasmid digestion to confirm.

Construction of BBa_K2275009

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8/13

  • PCR to get DNA of gudB from Pseudomonos putida and analyze by the DNA gel.
  • Digest gene glnA and pSB1C3-PLacI-sfGFP with HindⅢ and PstI. Analyze by the DNA gel
  • Ligase at 16℃, 4 hours and then transform into DH5α.

8/14

  • Select two colonies from the plates and pre-culturing.
  • Extract plasmid and use plasmid digestion to confirm.

Construction of BBa_K2275010

9/6

  • PCR to get rev-gudB.
  • Digest gene rev-gudB with XbaI and PstI and pSB1C3-PLacI-glnA with SpeI and PstI. Analyze by the DNA gel.
  • Ligase at 16℃ , 4 hours and then transform into DH5α

9/7

  • Select two colonies from the plates and pre-culturing.

9/8

  • Extract plasmid and confirm with plasmid digestion.

Construction of BBa_K2275011

9/13

  • Add a NsrR binding site to K381001 before RBS by PCR and analyze by DNA gel.

9/14

  • Digest linear PCR product with DpnI and self-ligate by phosphorylation
  • Transform the ligased product into DH5α and put at 37℃ for 12 hours.

9/15

  • Select two colonies from the plates and pre-culturing.

9/16

  • Extract plasmid and use plasmid digestion to confirm.

Construction of BBa_K2275012

9/13

  • Add a NsrR binding site to K381001 after RBS by PCR and analyze by DNA gel.

9/14

  • Digest the linear PCR product with DpnI and self-ligate by phosphorylation.
  • Transform the ligased product into DH5α and put at 37℃ for 12 hours

9/15

  • Select two colonies from the plates and pre-culturing.

9/16

  • Extract plasmid and use plasmid digestion to confirm.

Nitrate sensing test

7/1~7/31

  • Use M2 to quantitate fluorescence emitting by K381001 in lyophilized E. coli powder

8/1~8/31

  • Use our device to do the nitrate sensing test by K381001 in lyophilized E. coli powder.

Whole pathway test

10/7~10/10

  • Use our device and modified E. coli to test our whole project.