August

Milestones

9.Week

Organization

• We had a second radio interview in the Bielefeld university campus radio (radio 87.9 hertz) to introduce our project and the iGEM competition.
• Having an expert interview with Dr. Falk Harnisch from the Helmholtz Institute in Leipzig, who has got a great knowledge in the field of MFC.

MFC

Mediators

Cytochromes

• Restriction of ccmAH and psB1C3 and Suffix-Insertion
  • The backbone and shipping vector psB1C3 was cut with EcoRI, SpeI, the insert ccmAH with EcoRI and XbaI to perfom a standard suffix insertion.
  • We used approx. 200ng insert and 42ng vector, which repressnts a 2-fold molar excess of insert.
  • The Ligation was incubated for 15 min at 37°C, followed by a heat-inactivation at 80°C for 20 min.
  • The subsequent gel electrophoresis did not yield any positiv results, probably due to the use of buffer and enzymes from two different manufactures.

• Validation of all intermediate products via gel electrophoresis
  • A gel electrophoresis was performed to ensure that all former products have the correct size and this could be ruled out as an error source.
    • It showed that all products were indeed correct.

• Amplification of psB1C3 with insert-specific primers
  • The backbone and shipping plasmid psB1C3 was amplified with the new designed insert-specific primers for mtrCAB. These primers have a specific overlap complementary to 20nt at the beginning and end of the insert. This was necessary, because we had to deal with constant religation of the vector. We investigated this issue and found out, that prefix and suffix of the psB1C3 vector have a very similarity, which facilates religation. The correct band was extracted from the gel and cleaned up.

• • NanoDrop:
    • 4-0108-301: 1.1 ng/ul
    • 4-0108-302: 1.0 ng/ul

• Gibson-Assembly of psB1C3 and fragment 1,2 and 3 of the mtrCAB cluster
  • We use a custom 15 ul reaction mixture, prepared in our lab. After thawing on ice, a total amount of 5 ul DNA is added and the whole mixture is incubated at 50°C for approx. 60min.
  • Mixture:
  • psB1C3 (4-0108-301): 2.2 ng
  • Fragment1 (4-2607-304): 13.1 ng
  • Fragment2 (4-2707-004): 16.9 ng
  • Fragment3 (4-2607-301): 14.4 ng

• • Notes:The inserts were not equimolar and not added in a 3-fold excess to the vector

Transformation to self-made electrocompetent E.coli cells. A total volume of 1ul could be used.

Biosafety

• We did Gibson-Assembly with the different purified plasmids:
  • Fragment: "pSB1C3_plac_alr+ pSB1C3_alr_rev"+"pSB1C3_plac_pre+ pSB1C3_alr_suf",
  • "pSB1C3_alr_fwd+ pSB1C3_alr_rev"+"pSB1C3_alr_pre+ pSB1C3_alr_suf",
  • "araC_d1+ araC_d2"+"araC_d3+ araC_d4", with pKO4
  • After the Gibson Assembly we transformated the Gibson fragments into competent E.coli KRX cells and plated the cells on LB+CM20-plates

Porines

Week 15

Organization

• The TV station WDR made a video with us about our project. This can be seen in the

OWL local time, a television program of the WDR.

MFC

Mediators

Cytochromes

Biosafety

• We did plasmid isolation:
  • BBa_I13541 A: 95,1 ng/µL (9-88-451)
  • BBa_I13541 B: 165,1 ng/µL (9-88-452)
  • BBa_K914004 A white: 198,8 ng/µL (9-88-453)
  • BBa_K914004 B white: 204,9 ng/µL (9-88-454)
  • BBa_K914004 A red: 476,5 ng/µL (9-88-455)
  • BBa_K914004 B red: 400,9 ng/µL (9-88-456)
  • AraC_del A: 82,4 ng/µL (9-88-457)
  • AraC_del B: 64,5 ng/µL (9-88-458)
  • pSB1C3_alr A: 120,4 ng/µL (9-88-459)
  • pSB1C3_alr B: 168,4 ng/µL (9-88-460)
  • pSB1C3_alr C: 137,2 ng/µL (9-88-461)
  • ptac_alr A: 24,7ng/µL (9-108-451)
  • ptac_alr B: 39,6 ng/µL (9-108-452)

Porines

Week 16=

Organization

• Final preparations for CeBiTec pupil academy in the next week.

MFC

Mediators

Cytochromes

Biosafety

• We isolated the genome of Bacillus amyloliquefaciens
  • DMS-1061: 20ng/µL (9-138-451)
  • Wildtype H 10A1: 766,6 ng/µL (9-138-452)
  • Wildtype R+M+ 10A3: 825,2 ng/µL (9-138-453)

• We did restriktion analysis of the plasmids from 8.8.2013
  • BBa_I13541, BBa_K914004 white colony, BBa_K914004 red colony, araC, alr
  • Result: failed

• Colony-PCR:
  • BBa_I15341, BBa_K194004 white, BBa_K194004 red, pSB1C3_alr, ptac_alr+insertprimern
    
  • Result: failed

• Restriktionanalysis:
  • Alr 1,2,3,4
  • Result: no bands as expected

Porines

Week 17

Organization

• Our Team will participate at the conference ‘60 Years of DNA’ in Berlin at 14.

September.

MFC

Mediators

Cytochromes

Biosafety

• Amplification of DNase Ba:
  • Size: 350bp

Gel analysis of RNase Ba PCR.

Gel purification of the fragments:

• RNase Ba fwdrevinsert 27,5ng/µL (9-238-451)
• RNase Ba presufVektor 67,3ng/µL (9-238-452)

Porines