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 [1]. 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) [-1]. 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-neo^R
• ∆amyE::GP35-neo^R
• ∆mutS::beta-neo^R
• ∆mutS::GP35-neo^R

The four different strains where compared to each other to get compare there transformation efficienc in MAGE.

Methods

making beta and GP35

The beta CDS was codon optimized with JCat() and ordered from COMPANY. GP35 sequence was found from (GP35 article). The sequence was ordered from the same company as beta.

incertion of recombinases in the amyE locus of B. subtilis

Beta and GP35 was inserted into the plasmid known as pDG268neo by using PCR with G5 HF 2x master mix from NEB (primer sequence hyperlink?). The plasmid was transformed into E coli DH5alpha by heat chock and incubated over night. The plasmid was purified from E coli by using QiAPrep mini prep kit. Digesting with restriction enzymes validated the insertion. This plasmid integrates the CDS into the amyE locus in Bacillus subtilis. pDG268neo with ether beta or GP35 was linearized and transformed into B subtilis W168 using the CFrench protocol (make CFrench to hyperlink)

chromosomal DNA from B. subtilis 168 was isolated (protocol)

knock out mutS

The regions upstream and downstream of the mutS locus were amplified by PCR from the chromosomal DNA with primers that had overlap with GP35 and beta.

GP35 and beta were PCR amplified with primers that had overhangs that were homologies to the mutS locus in bacillus subtilis. GP35 and beta was assembled into the pSB1C3 plasmid by using gibson assembly and transformed into E coli and incubated over night. The plasmid was isolated by using mini prep.  The plasmids were verified using restriction enzymes.

beta and GP35 in pSB1C3 was linearized using XhoI, This also removed the Chloramphenicol gene from the plasmid. Beta and GP35 was transformed into B. subtilis.

Genomic DNA extraction (B. brevis)

Ligation of DNA

PCR -

• PCR
• Colony PCR
• Touch down
• …

Plasmid Purification Miniprep

Restriction enzyme digest (NEB)

Tyrocidine construct

Transformation and Preparation of E.coli
            -Preparation of Competent Cells
            -Transformation of Chemically Competent E.coli
Transformation of Bacillus

Optiminsing OGRE in Bacillus

Different amount of oligo was tested to optimize OGRE method for Bacillus subtilis.

The mismatch insertion frequency and oligo length was tested. The standard OGRE protocol was follow with the mismatches verified from 1-6 mismatches and oligoes from 50-100nt.

OGRE with 4 cycles were attempted every cycle to approximately 6 hours. This was done to test if the transformation efficiency would rise when more cycles were attempted

changing surfatin

The 5th A domain in the NRPS for surfactin was attempted changed from a aspartate to a asparagine. This was done by following the OGRE protocol using a oligo that would change the specificity from asp to asn.

Protocol

Timeline