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 protein substitution in the ribosomal S12 protein in B. subtilis 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 design using MODEST (Bonde et al., 2014). These oligos were successfully integrated into the two strains: ∆mutS::beta-neoR and ∆mutS::GP35-neoR.
All the strain were made by homologous recombineering. Plasmids containing cassettes that were able to do a double-crossover homologous recombineering 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 inserting 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 in a 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 was 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. About 500 bp up- and downstream from mutS was amplified, using primers with tails.
2. Recombinase and neoR expression cassettes was amplified from the pDG268neo_recombinase
3. Linearized pSB1C3 was used as template.
These fragments was 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 cells were prepared in following the protocol: Electroporation competent Bacillus subtilis 168. Electropration was carried out according to the MAGE in Bacillus subtilis 168 protocol. The colonies from the LB plates were colonies picked onto an LB and 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.Results
Results
| Total number of CFUs | Number of transformant | Transformation frequency | |
|---|---|---|---|
| ΔmutS::beta-neoR | 52 | 7 | 0.13 |
| ΔmutS::GP35-neoR | 100 | 1 | 0.01 |
Table 1. shows the data for the MAGE proof of concept.
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.
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 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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