Revision as of 17:06, 9 October 2022

pCT5c Type IIS Compatible Plasmid

Description

This plasmid is a mutated version of pCT5-bac 2.0 (Fig 1). The original plasmid was created by Claudia Schmidt-dannert lab and provided by Addgene (plasmid # 119872). pCT5-bac 2.0 has a novel p-isopropyl benzoate (cumate) inducible gene expression system, constructed by combining the strong constitutive Bacillus promoter P_veg with regulatory elements, CymR repressor, and CuO operator sequence. A sfGFP reporter was assembled under the cumate-inducible promoter for gene expression in Bacillus subtilis, B. megaterium, and Escherichia coli. This plasmid is known as a platform for both gram-positive and gram-negative expression host. Two antibiotic resistance was found, with ampicillin for E. coli and tetracycline for Bacillus strains. However, pCT5-bac 2.0 has three BsaI restriction sites at position 650, 2994, and 6475, making it incompatible with Type IIS assembly standards. During preliminary research at the beginning of the project, we realize there is limited shuttle vectors for Bacillus subtilis in the registry. Therefore, we have created a Type IIS compatible vector pCT5c (BBa_K4417000) using site directed mutagenesis (SDM) to remove all the forbidden sites (Fig 2).

Figure 1: pCT5-bac 2.0 containing three BsaI sites at position 650, 2994, and 6475

Figure 2: pCT5c (BBa_K4417000) with no BsaI or SapI site

For each BsaI restriction site, we have designed a set of SDM primers (BBa_K4417001, BBa_K4417002, BBa_K4417003, BBa_K4417004, BBa_K4417005, BBa_K4417006). The part was confirmed by sequencing and used in our further cloning.

Usage and Biology

• This part can be used as a shuttle vector for both B.subtilis and E.coli.
• Inducer: p-isopropyl benzoate (cumate).
• Cumate is non-toxic to the host. In our experiment, we tried to induce the GFP expression at 50 μM and 100 μM.
• The copy number of this plasmid in B.subtilis and E.coli is unknown.

Method

We used back-to-back orientation site directed mutagenesis to remove the forbidden BsaI sites, while the principle was to keep the amino acid sequence. Designed oligonucleotide primers conferred a desired mutation in a double-stranded DNA plasmid. BBa_K4417001 and BBa_K4417002 are the primer set for mutating the first BsaI site (SDM1). The nucleotide was mutated from c to g at position 647. BBa_K4417003 and BBa_K4417004 are the SDM primer for the second BsaI site, mutating c into g at position 6004 (SDM1,3). BBa_K4417005 and BBa_K4417006 are the third SDM primer, targeting at position 6471 to mutate t into c (pCT5c).

The protocol was:

• Assemble the following reagents in a thin-walled PCR tube.
  • 7.5 μL 2X PhusionMix
  • 1 μL 10 μM forward primer
  • 1 μL 10 μM reverse primer
  • 1 μL template DNA (1ng/μL)
  • 4.5 μL nuclease-free water
• Mix by pipetting up and down.
• Transfer to a thermocycler and perform the following cycling conditions with a temperature gradient.

• KLD treatment by assembling the following:
  • 1 μL PCR product
  • 5 μL 2X KLD reaction buffer
  • 1 μL 10X KLD enzyme mix
  • 3 μL nuclease-free water
• Transformation (Fig 3)

Figure 3: E. coli plates transformed with pCT5c (a) first round of SDM (SDM1) at BsaI site 1. (b) second round of SDM (SDM1,3) at BsaI site 1 and 3. (c) third round of SDM (pCT5c) at all BsaI sites.

After successful transformation, the plasmid was checked by diagnostic digest. 5 μL uncut and 10 μL cut samples from each round of SDM was loaded on a 1% agarose gel to check the band size. From Figure 4 it could be concluded that the site directed mutagenesis was successful and all the BsaI sites were removed. A new Type IIS compatible plasmid was created and could be used by other iGEM teams. BamHI and SacI enzyme was chosen to digest for the plasmid backbone. In lane 13, the upper band (6851bp) was gel extracted for ligation with part BBa_K4417009 and BBa_K4417010. The lower band at 1026bp, very faint but visible, was the sfGFP sequence.

Figure 4: Comparative gel for pCT5c site directed mutagenesis; 1: HyperLadder^TM 1kb, 2: pCT5-bac 2.0 uncut, 3: SDM1 uncut, 4: SDM1,3 uncut, 5: pCT5c uncut, 6: pCT5c cut with BsaI (3481bp, 2344bp, 2052bp), 7: SDM1 cut with BsaI (4396bp, 3481bp), 8: SDM1,3 cut with BsaI (7877bp), 9: pCT5c cut with BsaI, 10: pCT5-bac 2.0 cut with BamHI/SacI (6851bp, 1026bp), 11: SDM1 cut with BamHI/SacI (6851bp, 1026bp), 12: SDM1,3 cut with BamHI/SacI (6851bp, 1026bp), 13: pCT5c cut with BamHI/SacI (6851bp, 1026bp), 14: pCT5-bac 2.0 cut with BamHI/BsaI (3481bp, 2087bp, 2052bp, 257bp), 15: SDM1 cut with BamHI/BsaI (3481bp, 2309bp, 2087bp), 16: SDM1,3 cut with BamHI/BsaI (5790bp, 2087bp), 17: pCT5c cut with BamHI/BsaI, 18: HyperLadder^TM 1kb.

pCT5c plasmid was further confirmed by Sanger Sequencing. High-quality sequencing results were shown in Fig 5.

Figure 5:Sanger sequencing for pCT5c. (a) sequencing at the first BsaI site (b) sequencing at the second BsaI site (c) sequencing at the third BsaI site.

Characterization

Promoter function was evaluated by cumate testing.

Conclusion

After analysis the results, we were confident that the pCT5c was successfully mutated and compatible with Type IIS standard. Expected band sizes were shown from the diagnostic digest, and cumate testing provided validated results. Therefore, we chose to use this part as our shuttle vector in B.subtilis cloning.

References

1. Development of a synthetic cumate-inducible gene expression system for Bacillus. Seo SO, Schmidt-Dannert C. Appl Microbiol Biotechnol. 2018 Nov 3. pii: 10.1007/s00253-018-9485-4. doi: 10.1007/s00253-018-9485-4. 10.1007/s00253-018-9485-4 PubMed 30392122

2. Addgene plasmid # 119872; http://n2t.net/addgene:119872; RRID:Addgene_119872

Sequence and Features