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
<
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.