Part:BBa_K5177025
Proposed pTF_fimA 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 proposed fragment cassette of our pTF_fimA plasmid, which contains a 23 base-pair spacer sequence and homology arm sequences customised for the deletion of the fimA 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 in K12 MG1655 was labelled as “fimA” in NEB10b strain due to its high protein homology with 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 test it. We included the fragment cassette sequence of our pTF_fimA plasmid here as a potential part that future teams may find useful.
Our target cassettes, carrying the target gene spacer for sfmA (BBa_K5177024) or fimA (this part), 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 designed a potential target cassette for NEB5a fimA gene deletion that is compatible with the CRISPR-Cas12a system [1]. Below we provide the proposed modified fragment cassette sequence for the pTF_fimA plasmid, flanked by restriction sites for easy plasmid assembly.
Design
After realising that we did not delete the actual fimA gene from E. coli NEB5a, we also designed this new fragment cassette containing new custom spacer and homology arm sequences following the same method [1] and created the custom plasmid we label pTF_fimA.
Following the protocol of [1], the fimA 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 fimA spacer. Our custom dDNA was composed of the left and right homology arms of the fimA gene; the left homology arm was designed to be the 50 bp upstream of the gene’s 5’ end, including the start codon of fimA, while the right homology arm was designed to contain 50 bp of the gene’s 3’ end, including the last 9 codons.
Figure 1. Plasmid map for the proposed pTF_fimA plasmid and a close-up diagram of its fragment cassette. Our custom 23-bp fimA gene spacer, noted by “fimA spacer,” and dDNA sequence (homology arms), labelled “LHA” and “RHA,” are shown in both diagrams. The close-up diagram also depicts the SpeI and EcoRI restriction sites that can be used to clone the cassette into the pTF plasmid, much like our pTF_sfmA fragment cassette (
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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