Part:BBa_K5177024
pTF_sfmA plasmid fragment cassette
Summary
To create a strain of ΔsfmAΔfimA E. coli NEB5a, we sought to employ a CRISPR-Cas12a system [1] that can be easily adapted to be used for chromosomal gene deletion in E. coli. This system employs two plasmids: the first plasmid, pSIMcpf1 (Addgene ID: 153034), carries the Cas nuclease and the Lambda Red recombination genes. The second plasmid (referred to as pTF) expresses the guide RNA (gRNA) and carries the repair template, composed of two 50-bp homologous arms. Because of its modularity, we only needed to introduce our target gene spacer and donor DNA (dDNA) sequence into the pTF plasmid.
Here we provide the fragment cassette of our experimentally tested pTF_sfmA plasmid, which contains a 23 base-pair spacer sequence and homology arm sequences customised for the deletion of the sfmA gene in E. coli NEB5a. The fragment cassette is also flanked by restriction sites (for SpeI and EcoRI, respectively) for easy assembly. This fragment cassette can be cloned directly into the pTF plasmid of [1].
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal EcoRI site found at 346
Illegal SpeI site found at 1 - 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 346
Illegal SpeI site found at 1 - 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 346
Illegal BamHI site found at 81
Illegal XhoI site found at 90 - 23INCOMPATIBLE WITH RFC[23]Illegal EcoRI site found at 346
Illegal SpeI site found at 1 - 25INCOMPATIBLE WITH RFC[25]Illegal EcoRI site found at 346
Illegal SpeI site found at 1 - 1000COMPATIBLE WITH RFC[1000]
Background
The fimA gene (Ecocyc ID b0530) encodes the major type 1 pili structural subunit protein in E. coli NEB5a. As our project involves the expression of a heterologous pili monomer in E. coli NEB5a, we needed to prevent the expression of the native type 1 pili to ensure maximal production of our desired type IV e-pili, and therefore we aimed to chromosomally delete fimA in E. coli NEB5a.
To create this strain, we sought to employ a CRISPR-Cas12a system [1] that can be easily adapted to be used for chromosomal gene deletion in E. coli. This system employs two plasmids: the first plasmid, pSIMcpf1 (Addgene ID: 153034), carries the Cas nuclease and the Lambda Red recombination genes. The second plasmid (referred to as pTF) expresses the guide RNA (gRNA) and carries the repair template, composed of two 50-bp homologous arms. Because of its modularity, we only needed to introduce our target gene spacer and donor DNA (dDNA) sequence into the pTF plasmid.
Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and their associated proteins, Cas9 and Cas12a (Cpf1), among others, is a genome editing system often used due to its efficiency and ease of use [2]. CRISPR genome editing systems incorporate a gRNA that is integrated into a Cas protein. The gRNA directs the Cas protein to the edit site to produce a double-stranded break (DSB). The system then relies on the cell’s intrinsic homology-directed repair (HDR) mechanism to repair the DSB with provided dDNA [3].
Initially, we used E. coli NEB10b as our chassis of choice, as the ΔfimA version of this strain was already shown to be an effective chassis for type IV e-pili production [4]. Therefore, we designed our gene spacer and dDNA based on the NEB10b fimA gene (Biocyc ID: ECDH10B_RS02480). However, due to the discovery of intrinsic resistance of this strain to streptomycin that coincided with the resistance marker gene used in our target specific plasmid, we were faced with the choice of either changing the strain or to replace the resistance gene of our plasmid. We chose to change strains to E. coli NEB5a, as our protein annotation analysis showed that this strain appeared to carry a gene (Genbank ID: AOO68850.1) that encoded for the same fimbrial protein as the NEB10b fimA gene.
However, after the deletion of this gene was carried out in NEB5a, we became aware through genome analysis that NEB10b did not possess the K12 MG1655 fim operon and therefore did not have the K12 MG1655 fimA sequence; rather, the gene sequence normally labelled sfmA<i> in K12 MG1655 was labelled as “fimA” in NEB10b strain due to its high protein homology with <i>fimA. This led us to discover that E. coli K12 MG1655, and accordingly, the NEB5a strain, possessed multiple chaperone-usher operons homologous to the fim operon. The most highly expressed of these homologous operons in standard laboratory conditions was the sfm operon [5], which we already incidentally disrupted by removing sfmA. It has also been shown that the removal of any of these chaperone-usher operons provides benefits to cell growth and can potentially increase the biosafety of the strain [6]. Therefore, to capitalise on these benefits and achieve our original goal of preventing native pili expression, we aimed to create an E. coli NEB5a ΔfimAΔsfmA strain.
We designed a new plasmid, pTF_fimA, for the deletion of the bona fide fimA gene in our E. coli NEB5a ΔsfmA strain, but regrettably, we did not have enough time to experimentally use the pTF_fimA plasmid. We included the fragment cassette sequence of our pTF_fimA plasmid as a potential part (BBa_K5177025) that future teams may find useful.
Our target cassettes, carrying the target gene spacer for sfmA or fimA, and the respective donor DNA sequences, as well as restriction sites flanking the target specific cassette, both have the same total size of 351 bp and can be introduced into pTF via SpeI and EcoRI restriction sites. Here, we constructed and experimentally proved a customised pTF plasmid for sfmA gene deletion, which we named pTF_sfmA, as well as designed a potential target cassette for NEB5a fimA gene deletion that is compatible with the same CRISPR-Cas12a system. Below we provide the modified fragment cassette sequence of our pTF_sfmA plasmid as well as proposed pTF_fimA plasmid, flanked by restriction sites for easy plasmid assembly.
Design
As we were able to obtain the original pTF plasmid [1] to serve as our template, we designed primers containing the sfmA spacer and dDNA and used PCR followed by Gibson assembly to clone our desired sequences into the pTF plasmid and form our custom pTF_sfmA plasmid.
Following the protocol of [1], we designed our sfmA spacer and homology arms. First, the target gene was screened for Cas12 protospacer adjacent motifs (PAMs) with the sequence TTTV (where V is A, C, or G). We selected a PAM located in the middle of the gene and used the proceeding 23-bp sequence as sfmA spacer. Our custom dDNA was composed of the left and right homology arms of the sfmA gene; the left homology arm was designed to be the 50 bp upstream of the gene’s 5’ end, including the start codon of sfmA, while the right homology arm was designed to contain 50 bp of the gene’s 3’ end, including the last 9 codons.
After realising that we did not delete the actual fimA gene from NEB5a E. coli, we also designed a new custom spacer and homology arms following the same method as above. We have also provided this part (BBa_K5177025), but unfortunately we did not have enough time to experimentally confirm its effectiveness.
Figure 1. Plasmid maps of the pTF_sfmA plasmid, the pSIMcfp1 plasmid, and the pTF plasmid. Diagrams of the pSIMcfp1 plasmid and pTF plasmid were directly sourced from [1], and the pTF_sfmA plasmid map was created with BioRender.com. Our custom sfmA gene spacer, noted by “sfmA spacer,” and dDNA sequence (homology arms), labelled “LHA” and “RHA,” were cloned into the pTF plasmid. We used the pSIMcfp1 plasmid without modification.
Figure 2. Close-up diagram of the pTF_sfmA fragment cassette. Highlighted are the specific elements of the cassette. The 23-bp sfmA spacer as well as the left and right homology arms are indicated. Shown are also the SpeI and EcoRI restriction sites, which can be used to clone the cassette into the pTF plasmid through restriction enzyme-based cloning.
Figure 3. Oligonucleotide primers used in the customisation of the pTF plasmid for the deletion of the sfmA gene. [A] Overall positioning of the primers on the final plasmid, pTF_sfmA, [B] forward and reverse primers to introduce the sfmA spacer and the left homology arm, [C] forward and reverse primers to introduce the right homology arm and amplify the plasmid backbone.
Figure 4. Plasmid map for the proposed pTF_fimA plasmid and a close-up diagram of its fragment cassette (
Results
Using our customised pTF_sfmA plasmid in conjunction with the pSIMcfp1 plasmid, following the method described in [1], we were able to obtain an E. coli NEB5a ΔsfmA strain.
We confirmed sfmA deletion by colony PCR using primers that bind up- and downstream of the homologous arms in the E. coli NEB5a genome (Figure 5). A PCR product of 1205 bp indicates the presence of sfmA, while a PCR product of 692 bp indicates the successful deletion of sfmA.
Figure 5. Primers for colony PCR to confirm the deletion of sfmA. Created with Biorender.com
As showcased by Figure 6, the sfmA gene deletion efficiency was 50% (5/10 colonies screened). For one colony (lane 4 in Figure 2), a mixed genotype could be observed.
Figure 6. Colony PCR results of E. coli NEB5a following the protocol for chromosomal deletion of sfmA. Lane 1: 1 kb ladder, Lanes 2 to 11: colony PCR results for 10 colonies, Lane 12: negative control (resuspended cells were substituted with MQ H2O), Lane 13: positive control of E. coli NEB5a, Lanes 14 and 15: 1 kb ladder.
References:
[1] Jervis, A.J., Hanko, E.K.R., Dunstan, M.S., Robinson, C.J., Takano, E., Scrutton, N.S. A plasmid toolset for CRISPR‐mediated genome editing and CRISPRi gene regulation in Escherichia coli. Microbial Biotechnology. 2021 Mar 12;14(3):1120–9. Available from: https://enviromicro-journals.onlinelibrary.wiley.com/doi/10.1111/1751-7915.13780
[2] Asmamaw, M., Zawdie, B. Mechanism and Applications of CRISPR/Cas-9-Mediated Genome Editing. Biologics : Targets & Therapy [Internet]. 2021 Aug 21;15(1):353–61. Available from: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8388126/
[3] Ran, F.A., Hsu, P.D., Wright, J., Agarwala, V., Scott, D.A., Zhang, F. Genome engineering using the CRISPR-Cas9 system. Nature Protocols. 2013 Oct 24;8(11):2281–308. Available from: https://www.nature.com/articles/nprot.2013.143
[4] Ueki T., Walker, D.J.F., Woodard, T.L., Nevin, K.P., Nonnenmann, S.S., Lovley, D.R. An Escherichia coli Chassis for Production of Electrically Conductive Protein Nanowires. ACS Synthetic Biology. 2020 Mar 3;9(3):647–54. Available from: https://pubs.acs.org/doi/10.1021/acssynbio.9b00506
[5] Korea, C.G., Badouraly, R., Prevost, M.C., Ghigo, J.M., Beloin, C. Escherichia coli K-12 possesses multiple cryptic but functional chaperone-usher fimbriae with distinct surface specificities. Environmental Microbiology. 2010 Mar 23;12(7):1957–77. Available from: https://enviromicro-journals.onlinelibrary.wiley.com/doi/10.1111/j.1462-2920.2010.02202.x
[6] Qiao, J., Tan, X., Ren, H., Wu, Z., Hu, X., Wang, X. Construction of an Escherichia coli Strain Lacking Fimbriae by Deleting 64 Genes and Its Application for Efficient Production of Poly(3-Hydroxybutyrate) and l -Threonine. Applied and environmental microbiology [Internet]. 2021 May 26 [cited 2024 Apr 25];87(12). Available from: https://journals.asm.org/doi/10.1128/aem.00381-21
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