"
Team:Bielefeld-Germany/Labjournal/August
From 2013.igem.org
(Difference between revisions)
| Line 232: | Line 232: | ||
We did restriktion analysis of the plasmids from 8.8.2013<br> | We did restriktion analysis of the plasmids from 8.8.2013<br> | ||
BBa_I13541, BBa_K914004 white colony, BBa_K914004 red colony, araC, alr<br> | BBa_I13541, BBa_K914004 white colony, BBa_K914004 red colony, araC, alr<br> | ||
| - | Result: failed<br> | + | Result: failed |
| + | <br><br> | ||
| + | Colony-PCR: <br> | ||
| + | BBa_I15341, BBa_K194004 white, BBa_K194004 red, pSB1C3_alr, ptac_alr+insertprimern<br> | ||
| + | Result: failed | ||
| + | <br><br> | ||
| + | Restriktionanalysis:<br> | ||
| + | Alr 1,2,3,4<br> | ||
| + | Result: no bands as expected | ||
| + | <br><br> | ||
| + | |||
| + | |||
| Line 269: | Line 280: | ||
<h4><a href="https://2013.igem.org/Team:Bielefeld-Germany/BioSafety">Biosafety</a></h4> | <h4><a href="https://2013.igem.org/Team:Bielefeld-Germany/BioSafety">Biosafety</a></h4> | ||
<p></p> | <p></p> | ||
| + | <br><br> | ||
| + | Amplification of DNase Ba: | ||
| + | size: 350bp<br> | ||
| + | [[File:IGEM Bielefeld 2013 RNase Ba.png|200px|thumb|left|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) | ||
| + | |||
<h4><a href="https://2013.igem.org/Team:Bielefeld-Germany/Project/Porins">Porines</a></h4> | <h4><a href="https://2013.igem.org/Team:Bielefeld-Germany/Project/Porins">Porines</a></h4> | ||
Revision as of 19:18, 27 September 2013
August
Milestones
1.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
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.
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.
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
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
2.Week
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
3.Week
Organization
- Final preparations for CeBiTec pupil academy in the next week.
MFC
Mediators
Cytochromes
Biosafety
We isolated the genome of Bacillus amyloliquefaciensDMS-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
4.Week
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
[[File:IGEM Bielefeld 2013 RNase Ba.png|200px|thumb|left|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)
