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(1)pSB1C3-Pnox

① Expande the newly synthesized pGH-nox plasmid transformed glycerol stocks in 3mL LB media.

②Extract the plasmid from 3mL of the above bacteria suspension and elute with 30µL Elution buffer.

③Do restriction enzyme digestion of pGH-nox with the restriction enzymes EcoRI and PstI in 50µL system.

④Run DNA gel electrophoresis of the production of pGH-nox digestion in 3µL system. Confirm that the restriction enzymes digestion is successful. Get the product of enzyme digestion purified and then eluted by 30µL Elution buffer.

⑤Ligate the extractive together with new vector by ligase overnight in temperature of 16℃.

⑥Transform the ligation product into competent E. coli DH5α cells. After centrifugation, discard the supernatant, get the product suspended and concentrated into 100µL, then plate it onto a LB agar medium and incubate at 37°C overnight.

⑦After overnight incubation, inoculate five freshly grown single colony and incubate each with 1.5 mL LB medium overnight.

⑧Extract 5 tubes of plasmid from the above bacteria suspension and do restriction enzyme digestion of the plasmid with the restriction enzymes EcoRI and PstI in 10µL system. Then send them for sequencing.

⑧Extract 5 tubes of plasmid from the above bacteria suspension and do restriction enzyme digestion of the plasmid with the restriction enzymes EcoRI and PstI in 10µL system. Then send them for sequencing.

(2) pSB1C3-Pnox+RBS+BFP、pSB1C3-Pnox+RFP

①Do restriction enzyme digestion of existing plasmids pSB1C3- BFP, pSB1C3- RFP with the restriction enzymes EcoRI and XbaI.

②Do restriction enzyme digestion of the plasmid pGH-nox with the restriction enzymes EcoRI and SPeI.

③Ligate the extractive together with new vector (digested pSB1C3- BFP、pSB1C3- RFP) by ligase overnight.

④Transform the ligation product into competent E. coli DH5α cells. After centrifugation, discard the supernatant, get the product suspended and concentrated into 100µL, then plate it onto a LB agar medium and incubate at 37°C overnight.

⑤After overnight incubation, inoculate five freshly grown single colony and incubate each with 1.5 mL LB medium overnight.

⑥Extract 5 tubes of plasmid from the above bacteria suspension and do restriction enzyme digestion of the plasmid with the restriction enzymes EcoRI and PstI in 10µL system. Then send them for sequencing.

(3) The Monitoring of Fluorescence Protein

PSB1C3-nox is also induced into fluorescence without nicotine, indicating that nox was a non-inducible promoter.

(1)the Construction of the first version plasmids of nica（pMD-Pnica1 + RBS + GFP + ter，pMD/pSB1C3-Pnica2 + RBS + GFP + ter）

①Design the primer including predictive promoter and RBS, using pSB1C3-GFP plasmid as template, through the polymerase chain reaction amplifying the plasmids containing Pnica1+RBS+GFP+ter (69 ℃ annealing and 72 ℃ extending for 2 minutes) and Pnica2+RBS+GFP+ter (69 ℃ annealing and 72 ℃ extending for 2 minutes) .

②Purify the genetic segments of Pnica1+RBS+GFP+ter (whose length is about 1Kb) and Pnica2 + RBS + GFP + ter (whose length is about 1Kb).

③Ligate two genetic segments together with T-vector by T4 DNA ligase.

④Transform the ligation product into competent E. coli DH5α cells. After centrifugation, discard the supernatant, get the product suspended and concentrated into 100µL, then plate it onto a LB agar medium and incubate at 37°C overnight.

⑤After overnight incubation, inoculate three freshly grown single colony and incubate each with 3 mL LB medium overnight.

⑥Extract plasmids from the above bacteria suspension, each 3 tubes of 1.5mL, and do restriction enzyme digestion of the plasmids with the restriction enzymes EcoRI and PstI in 10µL system. Then send them for sequencing.

⑦.The result shows that pMD-Pnica1+RBS+GFP+ter-3 (nica1-3) and pMD-Pnica2+RBS+GFP+ter-1 (nica2-1) sequencing results matched the reference sequences (in which nica2-1 was reverse connected with T-vector).

⑧Using the above bacteria to detect the expression of green fluorescent protein of different colonies. The result as in figure 8, from which we indicated that Pnica1 was a nicotine non-inducible promoter. While the plasmid pMD-Pnica2+RBS+GFP+ter-1 transformed bacteria did not express GFP in the absence of nicotine, suggesting that Pnica2 might be a nicotine inducible promoter.

⑨ In order to further verify whether Pnica2 is a nicotine inducible promoter, we compounded a LB agar medium with nicotine concentration of 5g/L, and plated 10µL plasmid pMD-Pnica2-RBS-GFP-ter-1 transformed bacteria suspension (which is produce by bacterial glycerol stock overnight incubation) droplets on it, then incubate it overnight.

⑩Observe the medium under a fluorescence microscope and we found that there was weak green fluorescence. This result shows that nica2 is a nicotine inducible promoter as shown in the following picture.

⑪Do restriction enzyme digestion of the plasmid pMD-Pnica2 + RBS + GFP + ter with the restriction enzymes EcoRI and PstI in 50µL system.

⑫.Purify the genetic segment Pnica2 + RBS + GFP + ter (whose length is about 1kb) , and ligate the segments together with linear pSB1C3-vector by T4 DNA ligase. Successfully complete the construction of the plasmid pSB1C3-Pnica2 + RBS + GFP + ter.

(2) the construction of the second version of Nica series plasmid (pMD/pSB1C3-Pnica2+RBS+T7RNA poLymerase +PT7 +RBS +GFP+ter)

1.Design the primer including predictive promoter and RBS, using the plasmid pXS-Oxy4 (pSB1C3-Pvgb +RBS+T7RNA poLymerase+ PT7+RBS+GFP+ter) as template, through the polymerase chain reaction amplifying the genetic segment Pnica2+RBS+T7RNA poLymerase+PT7+RBS+GFP+ter.

2.Get the genetic segment Pnica2+RBS+T7RNA poLymerase+PT7+RBS+GFP+ter purified and store the product in single eppendorf tube.

3.Ligate the genetic segment Pnica2+RBS+T7RNA poLymerase+PT7+RBS+GFP+ter together with T-vector by T4 DNA ligase.

4.Transform the ligation product into competent E. coli DH5α cells. After centrifugation, discard the supernatant, get the product suspended and concentrated into 100µL, then plate it onto a LB agar medium and incubate at 37°C overnight.

⑤5.After overnight incubation, inoculate twenty four freshly grown single colony and incubate each with 3 mL LB medium overnight.

6.Using the primers T7poLy-F and the universal primer M13-F on the T vector, the PMD-Pnica2+RBS+T7RNA poLymerase+PT7+RBS+GFP+ter plasmid was used as a template PCR to verify whether the reverse insertion was performed. The size of the clone insert 21 does not match and should be about 4 kb. Therefore, the second method (directed insertion) is used to insert Pnica2+RBS+T7RNA poLymerase+PT7+RBS+GFP+ter into the T vector.

7.Extract plasmid from the above bacteria suspension with gene forward inserted plasmid pMD-Pnica2 + RBS + T7RNA poLymerase + PT7 + RBS + GFP + ter, and do restriction enzyme digestion of the plasmid with the restriction enzymes EcoRI and PstI in 50µL system.

10.Purify the genetic segments Pnica2+RBS+T7RNA poLymerase+PT7+RBS+GFP+ter and linear T-vector and ligate the two segments together by T4 DNA ligase overnight.

10.Transform the ligation product into competent E. coli DH5α cells. After centrifugation, discard the supernatant, get the product suspended and concentrated into 100µL, then plate it onto a LB agar medium and incubate at 37°C overnight.

10.After overnight incubation, the medium didn't form any bacteria strain. So we decide to take the third method into action:

ligate the genetic segment Pnica2+RBS+T7RNA poLymerase+PT7+RBS+GFP+ter together with pUC-19 vector (circular) by T4 DNA ligase.

11.Transform 1µL of the ligation product into competent E. coli DH5α cells. Plate 100µL of the transformation product onto a LB agar medium and incubate it at 37°C overnight.

12.After overnight incubation, inoculate four freshly grown single colony and incubate each with 2 mL LB medium overnight.