Revision as of 12:38, 12 October 2022

pCT5c Type IIS Compatible Plasmid

Description

pCT5c (BBa_K4417000) is a mutated version of pCT5-bac 2.0 (Fig 1). The original plasmid was created by Claudia Schmidt-Dannert lab (Addgene: #119872). pCT5-bac 2.0 has sfGFP under a 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. PCT5-bac2.0 is a shuttle vector for propagation and expression in Bacillus subtilis, B. megaterium, and Escherichia coli. Two antibiotic selections are used, with ampicillin under a E. Coli promotor and tetracycline under a Bacillus promotor. During preliminary research at the beginning of the project, we realized there was no functional vector for Bacillus subtilis in the registry. pCT5-bac 2.0 has three BsaI restriction sites at position 650, 2994, and 6475, making it incompatible with Type IIS (RFC 1000) assembly standards. Therefore, we have created a Type IIS compatible vector pCT5c (BBa_K4417000) using site directed mutagenesis (SDM) to remove all the forbidden sites from pCT5-bac2.0 (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 sfGFP expression with different cumate concentrations (10-100 μM).
• E. coli ori is a pMB1 derivative.
• B. sub ori is unknown.
• The copy number of this plasmid in B.subtilis and E.coli is unknown.
• Literature indicated this plasmid was synthesized from a previous plasmid, pCT5-bac 1.8, using Gibson assembly.

Method

We used back-to-back orientation site directed mutagenesis to remove the forbidden BsaI sites following NEB’s SDM protocol (https://international.neb.com/protocols/2013/01/26/q5-site-directed-mutagenesis-kit-protocol-e0554), while keeping the amino acid sequence in CDS regions. Designed oligonucleotide primers conferred a desired mutation in a double-stranded DNA plasmid.

BBa_K4417001 and BBa_K4417002 are the primers 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 pair for the second BsaI site, mutating c into g at position 6004 (SDM1,3).

Finally, BBa_K4417005 and BBa_K4417006 are the third SDM primer pair, targeting position 6471 to mutate t into c (pCT5c).

The protocol was:

• The following reagents were assembled 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
• Mixed by pipetting up and down.
• Transferred to a thermocycler to perform the following cycling conditions with a temperature gradient.

• Kinase, Ligase & DpnI (KLD) reaction protocol (https://international.neb.com/protocols/2013/01/26/q5-site-directed-mutagenesis-kit-protocol-e0554):
  • 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 3. We encountered some difficulties in using SDM primer pair 2, so we switched to BsaI site 3 first. After redesigning primer pair 2, we got (c) third round of SDM (pCT5c) at BsaI site 2.

After successful transformation, colonies were picked, grown and the plasmid isolated. The isolated plasmid was checked by diagnostic digest. 5 μL uncut and 10 μL cut samples from each round of SDM were 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 can be used by other iGEM teams. BamHI and SacI enzymes were chosen to digest the plasmid backbone to remove the GFP insert and to insert a transcriptional unit flanked by SapI prefix and suffix sited to make the construct TypeIIS compatible in line with the iGEM BioBrick standard. In lane 13, the upper band (6851bp) was gel extracted for ligation with parts BBa_K4417009 and BBa_K4417010. The lower band at 1026 bp, very faint but visible, was the sfGFP sequence that was removed.

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.

The mutations of the three BsaI sites in the pCT5c plasmid were further confirmed by Sanger Sequencing. High-quality sequencing results are shown in Fig 5.

Figure 5:Sanger sequencing for pCT5c. Top sequence is the original plasmid pCT5-bac 2.0, and the bottom sequence is our mutated pCT5c (a) sequencing at the first BsaI site (b) sequencing at the second BsaI site (c) sequencing at the third BsaI site.

Characterization

Cumate testing

Cumate promoter function was evaluated by cumate testing. 5 mL cultures of DH5-α transformed with pCT5c were induced with 50 µM and 100 µM cumate, and an un-induced control sample was included. Overexpression of sfGFP was visible by eye when collecting the cell pellets following 15hrs incubation with cumate at 37°C, as shown in Fig 6.

Figure 6:Cell pellets overexpressing sfGFP with cumate induction after 24hrs. The Colour change of the pellet from light brown to green was observed. Uninduced control cultures (-) do not express sfGFP, and no change in colour was seen.

Fluorescence measurements of GFP are shown in Figure 7. From a single culture, cells were incubated overnights and split into 5 parts, four of them were induced with different cumate concentrations (10 µM, 30 µM, 50 µM, and 70 µM) and one was not induced. Induced DH5-α pCT5 cultures were strongly fluorescent compared to the non-induced DH5-α pCT5 (~10-fold with 50 µM cumate and ~6-fold with 10 µM cumate).

Figure 7:Cumate testing in DH5-α transformed with pCT5c. Fluorescence reading from 0-10 hrs.

The growth rate of WT E. coli was measured at OD₉₀₀ as shown in Figure 8.

Figure 8:Growth curve of transformed E. coli in cumate testing.

pH Growth Curve for WT E. coli

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