Team:DTU-Denmark/Journal
Summary
For OGRE (Oligo Recombineering) to be effective there is two genetic improvements that can be done. The first and most important is that the organism need to express a recombinase protein such as the beta protein derived from the lambda RED E. coli phage. The second improvement is to destroy/inhibit the mismatch repair system [1]. In B. subtilis the genes mutS and mutL are taking care of the mismatch repair [2]. For proof of concept purposes deleting the mismatch repair system will be adequate. According to (REF) the protein encoded by mutL will not be functional without a functional mutS, this is why deletion of only mutS will be sufficient to take out the mismatch repair system. With respect to recombinase we will test two different recombinases: lambda-beta (codon optimized for B. subtilis 168) and gp35 (a recombinase from the B. subtilis phage SPP1) [3]. For duing proof of concept of OGRE in B. subtilis four different strains was producere via genetic recombineering. All derived from Bacillus subtilis 168:
- ∆amyE::beta-neoR
- ∆amyE::GP35-neoR
- ∆mutS::beta-neoR
- ∆mutS::GP35-neoR
The four different strains where compared to each other to get compare there transformation efficienc in MAGE.
Protocols
set gel into Electrophoresis chamber filled with 1X TAE buffer so the wells are covered.
mix 5 µl of your PCR reactions/sample with 1 µl 6X loading buffer.
pipette 5 µl into each well, pour 2-3 µl ladder in some so the size can be seen.
run at 140 V, for 30-40 min
A lot of lab work requires plates in order to produce results, and often you need a special concentration, a unique antibiotic mixed into the plates or a different composition than what is available at the lab you are in. If that is the case, the plate needs to be made from scratch, or somewhat along the way.
Protocol can be found her
Timeline
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Biology of the Future
2015-09-14
Our team member Pernille spent an afternoon giving a presentation on cell factories and synthetic biology to 30 highschool students. She says: "Synthetic biology is all about being creative and use your imagination to design bio-solutions that can impact the world. For that exact reason, it is neccesary to discuss which ethical values tat should drive research". The highschool students had lots of questions and opinion for the discussion about ethics.
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VWR to the rescue!
2015-09-01
We are happy to announce that the VWR is contributing our team with their awesome products!
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Visit from Fisher Scientific!
2015-08-26
Today we had a visit by Susanne Basse from Fisher Scientific. She came by our office with a wagon full of items, Thanks!
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Canoeing
2015-08-07
Today we took a day off from lab to go canoeing, followed by a barbeque at Chris' place.
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Wiki Wizard
2015-07-30
We have uploaded the first beta version of the Wiki Wizard to GitHub
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First BioBrick cloned into B. subtilis
2015-07-30
An expression cassette with lambda beta recombinase was sucesfully transformed into Bacillus subtilis. First step towards establishing MAGE in Bacillus subtilis completed!
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BioBrick High School Project
2015-07-23
3 high school students; Simran, Noor, and Charlotte, joined us and Biotech Academy for 5 days to make biosensors that changes color, when water is poluted. Read more at ADD and http://biotechacademy.dk.
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First sequencing results
2015-07-22
We received our first sequencing results. One step closer to our first BioBrick!
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Brevibacillus parabrevis arrives
2015-07-16
Brevibacillus parabrevis produces a lot of different antibiotics including tyrocidine. We will transfer the tyrocidine operon to our chassis Bacillus subtilis.
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We get our strain
2015-04-28
We get a plate of our chassis strain Bacillus subtilis W168.
We can now begin the work in the laboratory. -
BioBrick Workshop
2015-04-24
Southern University of Denmark and University of Copenhagen joined us for a three-day BioBrick tutorial, where we spent three days in the laboratory cloning, learning about iGEM, wiki design, and getting to know each other through social activities.
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First official team meeting
2015-01-14
Today we had our first official team meeting. We talked about the project and got to know each other.
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Introductory meeting
2014-11-25
This day, last years team held an introductory session about iGEM for interested people.
References
- Carr, P. A., Wang, H. H., Sterling, B., Isaacs, F. J., Lajoie, M. J., Xu, G., … Jacobson, J. M. (2012). Enhanced multiplex genome engineering through co-operative oligonucleotide co-selection. Nucleic Acids Research, 40(17). doi:10.1093/nar/gks455
- Ginetti, F., Perego, M., Albertini, A. M., & Galizzi, A. (1996). Bacillus subtilis mutS mutL operon: Identification, nucleotide sequence and mutagenesis. Microbiology, 142(8), 2021–2029. doi:10.1099/13500872-142-8-2021
- Sun, Z., Deng, A., Hu, T., Wu, J., Sun, Q., Bai, H., … Wen, T. (2015). A high-efficiency recombineering system with PCR-based ssDNA in Bacillus subtilis mediated by the native phage recombinase GP35. Applied Microbiology and Biotechnology, 99(12), 5151–5162. doi:10.1007/s00253-015-6485-5
Department of Systems Biology
Søltofts Plads 221
2800 Kgs. Lyngby
Denmark
P: +45 45 25 25 25
M: dtu-igem-2015@googlegroups.com