Team:DTU-Denmark/Journal
Summary
For MAGE (Multiplex Automated Genome Engineering) 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 MAGE 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
Methods
Gel electrophoresis
Genomic DNA extraction (B. brevis)
Gibson Assembly
Glycerol stock –preservation
Ligation of DNA
PCR -
- PCR
- Colony PCR
- Touch down
- …
Plasmid Purification Miniprep
Restriction enzyme digest (NEB)
Transformation and Preparation of E.coli
-Preparation of Competent Cells
-Transformation of Chemically Competent E.coli
Transformation of Bacillus
Timeline
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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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Contribution from Macrogen!
2015-08-20
We are pleased to announce that Macrogen is sponsoring our team with sequencing!
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Contributions from AKG!
2015-08-19
We are happy to announce that AKG Acoustics is sponsoring our team with headphones for presentations in Boston!
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Contributions from VWR - Bie & Berntsen A/S
2015-08-11
We are happy to announce that VWR - Bie & Berntsen A/S is sponsoring our team with lab consumeables!
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Contributions from Fisher Scientific
2015-08-11
We are happy to announce that Fisher Scientific is sponsoring our team with lab materials!
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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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Contribution from SnapGene
2015-08-03
We are happy to announce that the SnapGene is contributing our team with lisences to their awesome product!
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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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Contribution from the Lundbeck Foundation!
2015-07-15
We are happy to announce that the Lundbeck Foundation is contributing financially to our team!
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Contributions from In Vitro!
2015-05-06
We are happy to announce that In Vitro A/S is sponsoring our team with lab consumeables!
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Contributions from Frisnette
2015-04-30
We are happy to announce that Frisnette is sponsoring our team with lab consumeables!
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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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Contribution from the Otto Mønsted Fonden!
2015-03-08
We are happy to announce that the Otto Mønsted Fonden is contributing financially to our team!
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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.
