iGEM 2015 — Team NJAU_China

# Team:NJAU China/Notebook

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* PROJECT
  + [Description](https://2015.igem.org/Team%3ANJAU_China/Description)
  + [Experiments&Protocol](https://2015.igem.org/Team%3ANJAU_China/Experiments)
  + [Results](https://2015.igem.org/Team%3ANJAU_China/Results)
  + [Design](https://2015.igem.org/Team%3ANJAU_China/Design)
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* HUMAN PRACTICES
  + [Human Practice](https://2015.igem.org/Team%3ANJAU_China/Practices)
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  + [Safety](https://2015.igem.org/Team%3ANJAU_China/Safety)
* [MODEL](https://2015.igem.org/Team%3ANJAU_China/Modeling%20)
* [MEASUREMENT](https://2015.igem.org/Team%3ANJAU_China/Measurement)

Team:NJAU\_CHINA/Notebook

#### Here is our experimental process.Filled out by each of our experiment teammates. The progress contains the details of our experiment, including the results of each step and the failure. And progress in our experiment can be clearly understood through this program.

| DATE | LAB NOTE | RESULT |
| --- | --- | --- |
| 2015/3-4 | Discussed project | Wonderful |
| 2015/4/5 | Made DH5α competent cell | Succeed |
| 2015/4/8 | TransformedBBa\_R0082(Promoter (OmpR, positive)) | Succeed |
| 2015/4/10 | TransformedBBa\_K592018 (cph8 with strong RB S),BBa\_P0451(strongRBS:B0034+ cI (lambda): C0051+double terminator:B0010、B0012),BBa\_C0040 (tetracycline repressor from transposon Tn10 (+LVA)) | Succeed |
| 2015/4/15 | TransformedBBa\_K592016 (B0034-YF1-B0034-FixJ, blue light sensor and RR with RBS) , BBa\_K823006(constitutive promoter) and BBa\_K592006(FixK2 promoter) | Succeed |
| 2015/4/2-4/17 | Askedfor the cas9 part from other universities | Failed |
| 2015/4/10-18 | Askedfor the cas9 gene at our college and finally get from postgraduate | Succeed |
| 2015/4/20 | Made competent cell DH5α | Succeed |
| 2015/4/25 | TransformedBBa\_S03518 (B0034-TetR); | Succeed |
| 2015/4/28 | TransformedBBa\_S03877(Strong RBS+hol), BBa\_K081021(B003+Pcya+ terminator) | Succeed |
| 2015/4/29 | TransformedBBa\_B0015 (Double terminator，forward： B0010+B0012) | Succeed |
| 2015/5/2 | Got Hfr straIn; PCR cas9 sequence and attempted to ligated cas9 on pSB1C3 | Succeed |
| 2015/5/3 | Made DH5αcompetent cell | Succeed |
| 2015/5/4 | PCR tested the existence of Orit and TraI gene in Hfr; Double restriction digestedBBa\_S03877and BBa\_K081021；ligateddigestedBBa\_S03877 and digestedBBa\_K081021 | Succeed |
| 2015/5/5 | Transformed the product of ligation on the former day into DH5α( the successful product was marked as holpt) | Succeed |
| 2015/5/7-8 | T-A colony of OriT from Hfr | Failed |
| 2015/5/10 | Double restriction digestedholpt and BBa\_R0082;ligateddigestedholpt and BBa\_R0082 | Succeed |
| 2015/5/11 | Transformed the product of ligation on the former day into DH5α (the successful product was marked as holpto) | Succeed |
| 2015/5/11-12 | T-A colony of OriT from Hfr | Succeed |
| 2015/5/14 | Test the conjugation efficiency between Hfr and BL21 | Succeed |
| 2015/5/19 | Tired to ligated cas9 with constitutive promoter ;Double restriction digestedBBa\_S03518 and BBa\_B0015 ;ligateddigestedBBa\_S03518 and BBa\_B0015 | Ligation failed; digestedion succeed |
| 2015/5/20 | Transformed the product of ligation on the former day into DH5α (the successful product was marked as tetT) | Succeed |
| 2015/5/24 | T-A colony of TraI from Hfr ; Double restriction digestedBBa\_R0082 and holpto;ligateddigestedBBa\_R0082 and holpto | T-A colonyfailed; restriction digestedionsucceed |
| 2015/5/25 | Transformed the product of ligation on the former day into DH5α (the successful product was marked as cph8A) | Succeed |
| 2015/5/28 | Double restriction digestedtetT and BBa\_C0040;ligateddigestedtetT and BBa\_C0040 | Succeed |
| 2015/5/29 | Transformed the product of ligation on the former day into DH5α (the successful product was marked as tetra) | Succeed |
| 2015/5/9-6/1 | Overlap PCR to knock out TraI gene in Hfr | Failed |
| 2015/6/2 | T-A colony of TraI from Hfr; TransformedBBa\_I13504 (reporter 1.0RBS+GFP+Terminator) | T-A colony failed; Transform edation succeed |
| 2015/6/3 | Made DH5α competent cell | Succeed |
| 2015/6/4 | Double restriction digestedcph8A , tetra and BBa\_I13504;ligated digeste dcph8A and BBa\_I13504, , tetra and BBa\_I13504 | Succeed |
| 2015/6/5 | Transformed the product of ligation on the former day into DH5α (the successful product was marked as cpg and tetRg) | Succeed |
| 2015/6/9 | Double restriction digestedcph8A and tetRg;ligateddigestedcph8A and tetRg | Succeed |
| 2015/6/10 | Transformed the product of ligation on the former day into DH5α (the successful product was marked as ctg) | Succeed |
| 2015/6/12 | T-A colony of TraI from Hfr | Failed |
| 2015/6/19 | Double restriction digestedBBa\_K592016 (B0034-YF1-B0034-FixJ, blue light sensor and RR with RBS) and BBa\_B0015 (Double terminator，forward： B0010+B0012); ligateddigestedBBa\_K592016 and BBa\_B0015 | Succeed |
| 2015/6/20 | Transformed the product of ligation on the former day into DH5α (the successful product was marked as NT) | Succeed |
| 2015/6/20 | Made DH5αcompetent cell | Succeed |
| 2015/6/15-6/21 | DNA synthesis of promoter λ(the DNA is called Pci) | Succeed |
| 2015/6/25 | Double restriction digested NT,Pci , BBa\_I13504and BBa\_K592006;ligateddigested NT and BBa\_K592006, Pci and BBa\_K592006, Pci and BBa\_I13504 | Succeed |
| 2015/6/26 | Transformed the product of ligation on the former day into DH5α (the successful product was marked as NG,GCi and Cig) | Succeed |
| 2015/7/1 | Double restriction digested NG and BBa\_K823006;ligateddigested NG and BBa\_K823006 | Succeed |
| 2015/7/2 | Transformed the product of ligation on the former day into DH5α (the successful product was marked as cngg) | Succeed |
| 2015/7/4 | Transformed the plasmid pET28a into DH5α | Succeed |
| 2015/7/6 | Double restriction digestedpET28a and cngg；ligateddigestedpET28a and cngg | Succeed |
| 2015/7/7 | Transformed the product of ligation on the former day into DH5α (the successful product was marked as pETcngg) | Succeed |
| 2015/7/8 | Made BL21 competent cell | Succeed |
| 2015/7/10 | Transformed the plasmid pETcngg into Made BL21 | Succeed |
| 2015/7/13 | Double restriction digestedcpg and Pet28a;ligateddigestedcpg and Pet28a | Succeed |
| 2015/7/14 | Transformed the product of ligation on the former day into DH5α | Failed |
| 2015/7/15 | Made DH5αcompetent cell | Succeed |
| 2015/6/5-7/15 | TraIgene knock out in Genescript company | Succeed |
| 2015/7/17 | Transformed the product of ligation on the former day into DH5α | Failed |
| 2015/7/20 | Double restriction digestedcpg, ctg and BBa\_K823006 | Succeed |
| 2015/7/16-7/20 | Test the conjugation efficiency between Hfr and BL21 and between TraIknocked-out Hfr and DH5α | Succeed |
| 2015/7/21 | Transformed the product of ligation on the former day into DH5α | Failed |
| 2015/7/23 | Double restriction digestedcpg, ctg and BBa\_K823006 | Succeed |
| 2015/7/24 | Transformed the product of ligation on the former day into DH5α | Failed |
| 2015/7/25-8/4 | Synthetized “GFPsg+terminater+constitutive promoter+1.0RBS” sequence by company. | Succeed |
| 2015/8/5 | Made DH5αcompetent cell ; Ligatedconstitutive promoter with GFP | Succeed |
| 2015/8/7 | Twice single restriction digestedcpg, ctg and Pet28a;ligateddigestedcpg and Pet28a; ctg and Pet28a; Ligated cas9 on pMD-18T | Succeed |
| 2015/8/1-8/7 | Verified constructions of three plasmids needed for InterLab Study | Succeed |
| 2015/8/8 | Transformed the product of ligation on the former day into DH5α | Failed |
| 2015/8/10 | Triedto ligate “GFPsg+terminater+ constitutive promoter+1.0RBS” sequence with cas9 | Failed |
| 2015/8/10-8/12 | InterLab Study read florescence intensity: First attempt | Failed |
| 2015/8/13-8/15 | InterLab Study read florescence intensity: second attempt | Succeed |
| 2015/8/15 | Double restriction digested cpg and Pet28a;ligated digested cpg and Pet28a | Succeed |
| 2015/8/16 | Transformed the product of ligation on the former day into DH5α | Failed |
| 2015/8/20 | Ligatedcas9on pet-28a. | Failed |
| 2015/8/21 | Twice single restriction digested cpg, ctg and Pet28a;ligated digested cpg and Pet28a; ctg and Pet28a; Ligated cas9 on pMD-18T | Succeed |
| 2015/8/22 | Transformed the product of ligation on the former day into DH5α | Failed |
| 2015/8/21 | T-A colony of TraI from Hfr | Succeed |
| 2015/8/22 | Verification of T-A colony | Succeed |
| 2015/8/23 | DNA sequencing of traI | Failed |
| 2015/8/24-27 | T-A colony of TraI from Hfr again | Succeed |
| 2015/8/28 | Twice single restriction digested traI and pSB1C3; ligated traI and pSB1C3. | Failed |
| 2015/9/1-3 | Twice single restriction digested traI and pSB1C3; ligated traI and pSB1C3. | Succeed |
| 2015/9/4-7 | Functional verification of traI | Succeed |
| 2015/9/9 | Another T-A colony of OriT from Hfr | Succeed |
| 2015/9/10 | Made DH5αcompetent cell | Succeed |
| 2015/9/10 | Submit the biobrick | Succeed |