Difference between revisions of "Team:TJU China/Notebook/Group1"

Activate smURFP glycerol.

Figure 1. Incubate to activate the smURFP plasmid PCR amplification of smURFP fragments.

Figure 2. The result of nucleic acid gel electrophoresis of smURFP after PCR. Lane M, Marker. Lane1-8，smURFP.
Gel Extraction purification of the PCR product.
Find the arsenic ion plasmid inside the box and transform the plasmid into E.coli DH5α.

Transform the plasmid（Bba-J33201） inside the box.
Amplification of PCR fragments.
PCR amplification of arsenic ion loop.

Figure 3. The result of nucleic acid gel electrophoresis of Bba-J33201 after PCR. Lane M, Marker. Lane 1-6，BBa-J33201.

PCR amplification of ArsR promoter and ArsR protein fragment.
Transform plasmid pKM586 into E.coli BL21.
Activate and extract pKM586 plasmid.

Activate smURFP glycerol.

Figure 4. The result of nucleic acid gel electrophoresis after overlapping of ArsR Promoter and smURFP. Lane M, Marker. Lane 1-10, ArsR Promoter＋smURFP

Double digestion of pKM586 plasmid.

Figure 5. Double digestion of pKM586 with AatII and BamHI. Lane M, Marker. Lane 1，pKM586; Lane 2-3, plasmid pKM586 after double enzyme digestion
Overlap ArsR + Arsr Promoter,but failed.
Overlap ArsR Promoter + smURFP, failed.
Enzyme digestion of pwj66.

Enzyme digestion of pwj66.
Connect pwj66+ TARGETDNA.
Double digestion of pKM586.
Overlap ArsR + Arsr Promoter.
Overlap ArsR Promoter + smURFP.
PCR of ParsR promoter ArsR protein fragment.

Figure 6.LB media of pKM586 after transformed

Enzyme digestion of pwj66.
Connect pwj66+ spacer.
Double digestion pkm586.
Overlap ArsR + Arsr Promoter.
Overlap ArsR Promoter + smURFP.

Figure 7.The result of nucleic acid gel electrophoresis after overlapping of ArsR Promoter and smURFP. Lane M, Marker. Lane 1-7, ArsR Promoter＋smURFP. PCR of ParsR promoter ArsR protein fragment.

Anneal two J23104 oligo chains.
PCR amplification of Bba-J23104 fragments. .
Overlap J23104 + ArsR protein fragment.
Overlap smURFP + ArsR promoter.

Figure 8. The result of nucleic acid gel electrophoresis after overlapping of ArsR Promoter and smURFP. Lane M, Marker. Lane 1, smURFP. Lane 2-4, ArsR Promoter＋smURFP.

Overlapping the two overlapped fragments.
Gel Extraction purification.
Double enzyme digestion of the entire arsenic loop.
Double digestion of pKM586.

Figure 9.Double digestion of pKM586 with AatII and BamHI. Lane M, Marker. Lane 1,Plasmid pKM586. Lane 2, Plasmid pKM586 after double enzyme digestion
Connection the two fragments after enzyme digestion
Transform the product into E.coli DH5α

We did a full fragment overlap of the arsenic loop and the result failed. Double digestion of pKM586 vector, plasmid extraction results and double enzyme digestion results are not ideal

Figure 10.Double digestion of pKM586 with AatII and BamHI. Lane M, Marker. Lane 1, pKM586; Lane 2-3, plasmid pKM586 after double enzyme digestion

We modified the arsenic loop full-segment overlap system and the PCR program, and the results were successful.

Figure 11.The result of nucleic acid gel electrophoresis after overlapping the whole arsenic loop. Lane M, Marker. Lane1-7 the whole arsenic loop.

Reconstruction of pKM586 original plasmid, the quality of plasmid extraction is not high

Try pKM586 step-by-step double enzyme digestion pre-experiment, resulting in significant improvement in cutting efficiency. The arsenic loop complete fragment double digestion and pKM586 double digestion product gel recovery, as well as ligation transformation.

Figure 12.the arsenic loop after transformed into DH5α

The plasmid was extracted and verified by double enzyme digestion. The result was successful.

Figure 13.Double digestion to verify the ligation product. Lane M, Marker. Lane 1, Plasmid pKM586. Lane 2, Plasmid pKM586 single digestion with BamHI. Lane 3, Plasmid pKM586 double digestion with AatII and BamHI. Lane 4, Plasmid ArS. Lane 5, Plasmid ArS single digestion with BamHI. Lane 6, Plasmid ArS double gigestion with AatII and BamHI. Lane 7, Plasmid ArS. Lane 8, Plasmid ArS single digestion with BamHI. Lane 9, Plasmid ArS double digestion with AatII and BamHI.

Transform the successfully constructed plasmid into E. coli BL21, pick a single colony, incubate the tube for the first arsenic ion delivery experiment, but the result shows no fluorescence.

Figure 14.the arsenic loop after transformed into BL21

After the successful construction of the plasmid, we began to try to test whether the transformed E. coli can express fluorescence after adding arsenic ions. We chose to culture the tube for 6 hours and then add different concentrations of arsenic ions for detection. For the second time, we failed again.
And we continue to try to change the condition to make it express fluorescence.