Part:BBa_K1716000
Lambda Beta recombinase optimized for expression in B. subtilis
Recombination-mediated genetic engineering (recombineering) utlises homologous recombination to facilitate genetic modifications at any desired target by flanking the mutated sequence with homologous regions. Multiplex Automated Genome Engineering (MAGE) is a method for rapid and efficient targeted programming and evolution of cells through cyclical recombineering using multiple single-stranded DNA oligonucleotides (oligos). The MAGE protocol utilises the λ Red recombination system in combination with an (temporary) inactivation of the mismatch repair system and consists of 7 steps that can be done with standard laboratory equipment (Wang, 2009). As MAGE utilises oligos, only the Beta protein of the λ Red system is required. This BioBrick encodes an optimised coding sequence for lambda phage derived beta recombinase. When expressed in Bacillus subtilis, oligo recombineering efficiencies were increased. [http://2015.igem.org/Team:DTU-Denmark/Project/MAGE Please see our project page].
Quick Guide: How to use
1. Express lambda beta in B. subtilis (or another organisms) by insertion into must (mismatch repair) gene. Read more about how we did that using standard B. subtilis BioBricks available from the registry under our project [http://2015.igem.org/Team:DTU-Denmark/Project/MAGE here]. 2. Make electro/MAGE-competent B. subtilis cells. Find our protocol here. 3. Design your 90-mer oligo. If you want to design oligos targeting NRPS use our NRPS Oligo Designer (NOD). Otherwise we suggest using [http://modest.biosustain.dtu.dk MODEST]. You can also read more about how to to multiplex engineering to design oligo chips [http://2015.igem.org/Team:DTU-Denmark/Project/Background here] for low-cost library generation. 4. Electroporate the cells. Read more about our optimisation of MAGE in Bacillus subtilis and cos-MAGE for higher efficiencies [http://2015.igem.org/Team:DTU-Denmark/Project/POC_MAGE here].
Methods and Experimental Design
Four Bacillus subtilis strains which expressed a recombinase were created by genetically engineering the wild type strain 168:
- ∆amyE::beta-neoR
- ∆amyE::GP35-neoR
- ∆mutS::beta-neoR
- ∆mutS::GP35-neoR
For proof of concept we decided to make a single point mutation in the ribosomal S12 protein in B. subtilis, which results in resistance to the antibiotic streptomycin. The required change is a lysine to arginine substitution at position 56 of the protein. The S12 subunit is coded by the rpsL gene (Barnard et al. 2010). An oligo that could integrate this change was designed using MODEST (Bonde et al., 2014). These oligos were successfully integrated into the two strains: ∆mutS::beta-neoR and ∆mutS::GP35-neoR.
All of the strains were made by homologous recombination. Plasmids containing cassettes that were able to do a double-crossover homologous recombination into the genome of B. subtilis 168 (referred to as knockout (KO) plasmid). These were used to, simultaneously, delete the desired gene (amyE or mutS) and insert the expression cassette for one of the recombinases: beta or GP35.
Figure 1. Shows the general concepts of the two plasmids pDG268neo_recombinase and pSB1C3_recombinase. Both exist in two versions, one with each of the recombinase proteins' CDSs (Beta and GP35). They also have different RBSs since they are optimized for the CDS. Upstream of neoR is a promoter and RBS and downstream of neoR is a terminator, but sequences and positions of these features are not known.
Four different plasmids were assembled to make the four MAGE ready strains:
pDG268neo_Beta-neoR
pDG268neo_GP35-neoR
pSB1C3_Beta-neoR
pSB1C3_GP35-neoR
Insert of recombinase in amyE
For pDG268neo_recobinase a DNA sequence containing following features
● Promoter: PliaG from BBa_K823002 was used.
● RBSs were optimized for the specific CDS using the salis lab RBS calculator (https://www.denovodna.com/software/)
● CDS for recombination protein beta or GP35
● Terminator: we use rho-independent Part:BBa_B0014
Sequence was ordered from IDT as two gblocks (for each recombinase) with overlapping regions, thus they can be assembled with Gibson assembly.
pDG268neo was linearized by PCR with primers, so that the native lacZ was omitted. This linearized plasmid was purified and used in a Gibson assembly reaction with two cognate gblocks.
Figure 2. Gibson assembly of the pDG268neo_GP35, the gblocks were fused to the “gp35 Gblocks fused” in the same reaction. The Gibson assembly of pDG268neo_beta was similar.
This resulted in two different plasmids pDG268neo_beta and pDG268neo_gp35.
mutS deletion
The two mutS KO plasmids was made of the following DNA fragments:
1. Flanking regions upstream and downstream of mutS from B. subtilis genome was amplified to generate the homologous regions for homologous recombination and deletion of mutS.
2. Recombinase and neoR expression cassettes were amplified from the pDG268neo_recombinase
3. Linearized pSB1C3 was used as template.
These fragments were assembled into two different plasmids pSB1C3_mutS::beta-neoR and pSB1C3_mutS::gp35-neoR using Gibson assembly.
Figure 3. Gibson assembly of the pSB1C3_beta. The feature called “mutL” corresponding mutS downstream. The Gibson assembly of pSB1C3_GP35 was similar.
All plasmids were verified by restriction enzyme digestion.
We prepared naturally competent B. subtilis 168 to be transformed. The plasmids were linearized with restriction enzymes. Then transformed into naturally competent B. subtilis 168, transformants were selected on 5ɣ neo. Transformants were verified with colony PCRs.
Electroporation competent Bacillus subtilis 168. was electroplated with MAGE oligos and plated on nonselective plates. The colonies from the LB plates were transferred to LB + 500y streptomycin (strep) plates. Plates were incubated at 37 degC for 48 hours and CFUs were counted on both the LB and the strep plates in order to determine recombination frequencies.
Results
| Total number of CFUs | Number of colonies on antibiotic plates | Recombineering frequency | |
|---|---|---|---|
| ΔmutS::beta-neoR | 52 | 7 | 0.13 |
| ΔmutS::GP35-neoR | 100 | 1 | 0.01 |
Table 1. shows the data for the MAGE comparing efficiencies between GP35 and Lambda Beta.
Second replication of the plates:
Figure 4. Pictures shows the first and second replication of the transformants. From left to right of ΔmutS::GP35-neoR replicate1, ΔmutS::GP35-neoR replicate2 , ΔmutS::beta-neoR replica1, and ΔmutS::beta-neoR replica2.
Conclusion
Recombineering efficiencies were higher, when Lambda Beta was used as recombinase than GP35. Previously, the opposite was concluded by Sun et al. that GP35. They used long ssDNA oligo of >1,000 nucleotides, which were generated by PCR. We used oligos with an length of around 90 nucleotides. This shows that the length of the optimal oligo depends on recombinase used. For our system, we wish to use Chip-based oligos. For that purpose long oligos are not suitable making lambda beta more applicable for our system. We do notice though that Sun et al. reach very high recombineering efficiencies which may be attributed to the long ssDNA used. B. subtilis has longer Okazaki fragments than E. coli and S. cerevisae.
References
Barnard, A. M. L., Simpson, N. J. L., Lilley, K. S., & Salmond, G. P. C. (2010). Mutations in rpsL that confer streptomycin resistance show pleiotropic effects on virulence and the production of a carbapenem antibiotic in Erwinia carotovora. Microbiology, 156(4), 1030–1039. Bonde, M. T., Klausen, M. S., Anderson, M. V., Wallin, A. I. N., Wang, H. H., & Sommer, M. O. A. (2014). MODEST: a web-based design tool for oligonucleotide-mediated genome engineering and recombineering. Nucleic Acids Research, 42(W1), W408–W415. doi:10.1093/nar/gku428 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 Wang, H. H., Isaacs, F. J., Carr, P. A., Sun, Z. Z., Xu, G., Forest, C. R., & Church, G. M. (2009). Programming cells by multiplex genome engineering and accelerated evolution. Nature, 460(7257), 894–898. doi:10.1038/nature08187 Photo credit go MAGE cycle: Michael Schantz Klausen
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 245
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