iGEM 2015 — Team Hong_Kong-CUHK

# Team:Hong Kong-CUHK/Notebook

* [HOME |](https://2015.igem.org/Team%3AHong_Kong-CUHK)
* ABOUT |

  + [Our Team](https://2015.igem.org/Team%3AHong_Kong-CUHK/Attributions)
  + [Attributions](https://2015.igem.org/Team%3AHong_Kong-CUHK/Sponsor)
* PROJECT |

  + [Magnetosome](https://2015.igem.org/Team%3AHong_Kong-CUHK/Team)
  + [The insertion kit](https://2015.igem.org/Team%3AHong_Kong-CUHK/insertion_kit)
  + [Experiments & Methods](https://2015.igem.org/Team%3AHong_Kong-CUHK/methods)
  + [Results](https://2015.igem.org/Team%3AHong_Kong-CUHK/Results)
  + [Part Improvement](https://2015.igem.org/Team%3AHong_Kong-CUHK/Description)
  + [Application](https://2015.igem.org/Team%3AHong_Kong-CUHK/Design)
  + [Modeling](https://2015.igem.org/Team%3AHong_Kong-CUHK/Modeling)
* [PARTS |](https://2015.igem.org/Team%3AHong_Kong-CUHK/Parts)
* [NOTEBOOK |](https://2015.igem.org/Team%3AHong_Kong-CUHK/Notebook)

  + [Wet Lab Journal](https://2015.igem.org/Team%3AHong_Kong-CUHK/Notebook)
  + [Protocols](https://2015.igem.org/Team%3AHong_Kong-CUHK/Experiments)
* [SAFETY |](https://2015.igem.org/Team%3AHong_Kong-CUHK/Safety)
* [POLICY & PRACTICE |](https://2015.igem.org/Team%3AHong_Kong-CUHK/Practices)

  + [Questionnaire](https://2015.igem.org/Team%3AHong_Kong-CUHK/Questionnaire)
  + [International Questionnaire](https://2015.igem.org/Team%3AHong_Kong-CUHK/iQuestionnaire)
* [COLLABORATIONS |](https://2015.igem.org/Team%3AHong_Kong-CUHK/Collaborations)
* [iGEM](https://igem.org/Main_Page)
* [![CUHK](https://static.igem.org/mediawiki/2015/0/02/CUHK_Logo.png)](http://www.cuhk.edu.hk/english/index.html)
* ![CUHK iGEM Team 2015](https://static.igem.org/mediawiki/2015/c/c7/CUHK_iGEM_Team_logo.png)

Wet Lab Journal

March

1. Learning Lab techniques and protocols
2. Brainstorming ideas
3. Researches

April

1. Project decided
2. Preparation of special medium and plate for *A. vinelandii*
3. Study the growth curve characteristics of *A. vinelandii*
4. Preparation of Competent Cells of dh5α and BL21, aliquot the competent cells, stored in liquid nitrogen and then -80C refrigerator.
5. Test the transformation efficiency of Competent Cells.
6. Make plate with antibiotics -Ampicillin, kanamycin and chloramphenicol.
7. Make LB brooth

May

1. Purchase for primers
2. Primer dilution of the all arrived primers
3. PCR flanking sequence of *mamAB* operons (main part of BBa\_K1648000) from *Magnetospirillum gryphiswaldense*(MSR-1), following by gel electrophoresis.
4. Restriction of PCR product flanking sequence of *mamAB* operons, ligase into C backbone and transform into dh5α, forming BBa\_K1648000
5. PCR flanking sequence of *mamXY*, *mamGFDC* and *mms6* operons (main part of BBa\_K1648001) from *Magnetospirillum gryphiswaldense*(MSR-1), following by gel electrophoresis.
6. Restriction of PCR product flanking sequence of *mamXY*, *mamGFDC* and *mms6* operons, ligase into C backbone and transform into dh5α, forming BBa\_K1648001
7. Sending BBa\_K1648000 and BBa\_K1648001 to sequencing
8. Test the transformation efficiency of Competent Cells
9. Make plate with antibiotics -Ampicillin, kanamycin and chloramphenicol
10. Make LB brooth

June

1. Restriction of BBa\_K1648000 with BBa\_J13002 in the front and BBa\_B0015 at the back, ligase into C backbone and transform into dh5α, forming BBa\_K1648002
2. Restriction of BBa\_K1648000 with BBa\_J13002 in the front and BBa\_B0015 at the back, ligase into C backbone and transform into dh5α, forming BBa\_K1648003
3. Sending BBa\_K1648002 and BBa\_K1648003 to sequencing
4. Make LB plate with antibiotics -Ampicillin, kanamycin and chloramphenicol
5. Make LB brooth

July

1. PCR *mamHIEJKLMN*, *mamOPAQRBSTU*, *mamXY*, *mamGFDC* and *mms6* operons from *Magnetospirillum gryphiswaldense*(MSR-1), following by gel electrophoresis
2. Preparation of Competent Cells of dh5α and BL21, aliquot the competent cells, stored in liquid nitrogen and then -80C refrigerator
3. Make LB plates with antibiotics -Ampicillin, kanamycin and chloramphenicol
4. Make LB brooth

August

1. Preparation of Competent Cells of *A. vinelandii*
2. Transformation of linearized BBa\_K1648002 into *A. vinelandii*
3. Screening of the transformed *A. vinelandii* by colony PCR
4. Overlapping PCR of Insertion kit (precursor of BBa\_K1648004)
5. Restriction digestion of Overlapping PCR product of Insertion kit, ligase into circular insertion kit and transform into dh5α, forming BBa\_K1648004
6. Arrival of synthetic sequence of GFP-nanobody
7. Restriction of synthetic sequence of GFP-nanobody, ligase into C backbone and transform into dh5α, forming BBa\_K1648005
8. Restriction of BBa\_K1648005, ligase into BBa\_K1648004 and transform into dh5α, forming BBa\_K1648006
9. Sending BBa\_K1648004, BBa\_K1648005, BBa\_K1648006 to sequencing
10. Mutated *oprF* is synthesized by IDT and make the PCR product of *oprF* from *Azotobacter vinelandii*
11. Make LB, B, BN, BN-MO plates with antibiotics -Ampicillin, kanamycin and chloramphenicol
12. Make LB brooth and BN medium

September

1. Introducing PCR product of *mamHIEJKLMN* into transformed *A. vinelandii*
2. Screening of the homologous-recombinant *A. vinelandii*
3. Restriction of PCR product of *oprF* and pSB1C3, ligase into C backbone and transform into dh5α, forming BBa\_K1648045
4. Restriction of PCR product of *oprF* with BBa\_J13002 in the front, ligase into A backbone and transform into dh5α, forming BBa\_K1648048
5. Restriction of synthesized mutated *oprF* with BBa\_R0040 in the front, ligase into C backbone and transform into dh5α, forming BBa\_K1648049
6. Restriction of BBa\_k1172501 with BBa\_J13002 in the front, ligase into C backbone and transform into dh5α, forming BBa\_K1648050
7. Make LB, B, BN, BN-MO plates with antibiotics -Ampicillin, kanamycin and chloramphenicol
8. Make LB brooth and BN medium
9. Submission of all biobricks to the registry