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Article

A Toolkit for Effective and Successive Genome Engineering of Escherichia coli

by
Bahareh Arab
1,
Adam Westbrook
1,2,
Murray Moo-Young
1 and
Chih-Hsiung Perry Chou
1,*
1
Department of Chemical Engineering, University of Waterloo, 200 University Avenue West, Waterloo, ON N2L 3G1, Canada
2
Genecis Bioindustries Inc., 633 Coronation Drive Unit 10, Scarborough, ON M1E 2K4, Canada
*
Author to whom correspondence should be addressed.
Fermentation 2023, 9(1), 14; https://doi.org/10.3390/fermentation9010014
Submission received: 14 November 2022 / Revised: 12 December 2022 / Accepted: 20 December 2022 / Published: 23 December 2022
(This article belongs to the Special Issue Bioprocess and Metabolic Engineering)
Browse Figures
Versions Notes

Abstract

:
The bacterium Escherichia coli has been well-justified as an effective workhorse for industrial applications. In this study, we developed a toolkit for flexible genome engineering of this microorganism, including site-specific insertion of heterologous genes and inactivation of endogenous genes, such that bacterial hosts can be effectively engineered for biomanufacturing. We first constructed a base strain by genomic implementation of the cas9 and λRed recombineering genes. Then, we constructed plasmids for expressing gRNA, DNA cargo, and the Vibrio cholerae Tn6677 transposon and type I-F CRISPR-Cas machinery. Genomic insertion of a DNA cargo up to 5.5 kb was conducted using a transposon-associated CRISPR-Cas system, whereas gene inactivation was mediated by a classic CRISPR-Cas9 system coupled with λRed recombineering. With this toolkit, we can exploit the synergistic functions of CRISPR-Cas, λRed recombineering, and Tn6677 transposon for successive genomic manipulations. As a demonstration, we used the developed toolkit to derive a plasmid-free strain for heterologous production of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) by genomic knock-in and knockout of several key genes with high editing efficiencies.

1. Introduction

Over the past decades, bacterium E. coli has been exploited as a workhorse for industrial applications [1], which often require the host strain to be genetically modified to develop proper phenotypes. Basically, genetic modification is conducted to change (i.e., increase, decrease or wipe out) the expression levels of key genes, either native or foreign ones. Traditionally, enhancing (or reducing) gene expression relies on the use of vector systems (e.g., plasmid or virus), whereas inactivating gene expression is conducted by targeting endogenous genes on the host genome for mutation (i.e., gene knockout). However, the presence of extrachromosomal plasmids containing foreign genes can adversely impact cell physiology [2,3]. On the other hand, genomic integration of these foreign genes can enhance cell fitness and genetic stability [4], eliminating the necessity of using antibiotics to maintain plasmid stability [5]. While extrachromosomal plasmid systems appear to be relatively easy for implementation, targeting host genome for manipulation remains challenging and it is not even common until recently when novel biotechnologies for site-specific genome engineering become available.
Various genome engineering technologies were developed in early days based on (1) random mutagenesis via strain exposure to UV radiation or chemical mutagens followed by phenotype screening [6,7], (2) zinc-finger nuclease (ZFN) [8] and transcription activator-like effectors (TALEs) mutagenesis [9], (3) base editing [10] and prime editing [11], (4) genetic/genome recombination [12] and Multiplex automated genome engineering (MAGE) [13], (5) transposon mutagenesis [14], (6) phage transduction [15], and bacterial conjugation [16]. However, these traditional technologies are more limited to gene knockout and have various disadvantages, such as non-specific and non-reproducible mutations, hard-to-characterize mutations/mutants, labor-intensive and time-consuming protocols, etc. In early 21st century, technologies for site-specific genome engineering started to be developed, enabling targeted inactivation of endogenous genes in E. coli, and recombineering was one of them. Basically, recombineering relies on homologous recombination, which is mediated by phage recombinases such as RecET from Rac prophage or λRed proteins (i.e., Gam, Beta, Exo) [17], to integrate a double-stranded and single-stranded DNA fragment into the host genome [18]. In the λRed machinery, Gam prevents the degradation of the foreign DNA [19], Exo (lambda exonuclease) generates single-stranded 3′ overhangs, and Beta mediates annealing of homology arms to the complementary DNA target in the genome [20] (Figure 1A). Recombineering has been extensively applied to inactivate endogenous genes and a comprehensive E. coli mutant library containing the single mutations to all non-essential genes, i.e., Keio collection, was constructed [21], significantly facilitating derivation of E. coli gene-knockout mutants. On the other hand, recombineering has major technological drawbacks, such as low recombination efficiencies, especially upon integrating large DNA cargos, and requiring multi-step processes [4,13,22], limiting its primary application to gene knockout.
More recently, site-specific genome engineering based on the clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated proteins (Cas) was developed. The CRISPR-Cas system is a well-known machinery that reinforces immunity in bacteria and archaea by eradicating the invading foreign genetic elements [23,24]. Basically, the CRISPR-Cas system employs sequence-specific protospacers that are expressed as CRISPR RNAs (crRNAs) to guide Cas nucleases to the DNA/RNA target for degradation through the recognition of the protospacer-adjacent motif (PAM) downstream of the targeted site [25,26]. This natural defending mechanism in RNA-guided DNA cleavage has been repurposed as a powerful toolkit for genome engineering, particularly site-directed mutagenesis, in both prokaryotes and eukaryotes [27], successfully generating engineered cells for a wide range of applications [28] such as therapeutics [29] and engineered food crops [30].
Even with successful application to gene knockout and site-directed mutagenesis for genome editing, the CRISPR-Cas system has a limited editing efficiency upon inserting DNA cargos, particularly large ones, into the genome. The issue can be complemented by the new transposon-associated CRISPR-Cas system. Recent studies demonstrated the effective synergy between Vibrio cholerae Tn6677 transposon and type I-F CRISPR-Cas system for site-specific DNA transposition in E. coli [31,32,33]. Basically, Vibrio cholerae Cascade (VcCascade), encoded by Vibrio cholerae Tn6677, consists of Cas6, Cas7, and naturally fused Cas8-Cas5 (simply Cas8) protein subunits [34]. The hairpin on the 3′ end of crRNA binds to Cas6, while the 5′ end binds to Cas8 which is responsible for PAM site recognition and R-loop formation [35]. The transposition machinery comprises the TniQ subunit and transposition proteins of TnsA, TnsB, and TnsC. The TniQ homodimer is bound to the Cas6 protein of VcCascade to form the VcCascade-crRNA-TniQ complex [36]. To insert the DNA cargo, the crRNA directs the VcCascade-crRNA-TniQ complex to the targeted locus of the genome. TniQ communicates with TnsC to mediate the transposition by employing TnsAB to excise the DNA from its donor plasmid, recognizing and binding to the transposon right- (TR) and left- (TL) end sequences of the DNA cassette, and integrating it into the targeted locus [35,37] (Figure 1B). Unlike most transposons that randomly insert the DNA cargo into the genome, the Tn6677 transposon associated with the CRISPR-Cas system precisely inserts the DNA cargo at a specified distance downstream of the targeted locus [38], i.e., approximately 50 base pairs downstream of the 3′ end of the targeted site in either left to right or right to left orientation [31,39]. In addition to the programmability, this system can insert kilobase-pair-sized DNA cargos with enhanced efficiency and accuracy, making it a promising molecular toolkit for large-scale and pathway-sized DNA integration into the genome [22,40]. However, the transposon-associated CRISPR-Cas system is unsuitable to perform gene deletion or site-directed mutagenesis.
In this study, by integrating the three biomolecular mechanisms of CRISPR-Cas, Tn6677 transposon, and recombineering, we developed a programmable and efficient toolkit for extensive genome editing, including both site-specific inactivation of endogenous genes (gene knockout) and insertion of large DNA cargos (gene knock-in). The toolkit contains key engineered E. coli strains for convenient implementation of multiple rounds of genome editing and each editing round is based on one-step transformation with high editing efficiencies, enabling customized derivation of plasmid-free E. coli strains for industrial applications. As a demonstration, we used the developed toolkit to derive a plasmid-free engineered strain for the production of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) through metabolically implementing a heterologous PHBV-biosynthetic pathway (Figure 2) and then redirecting dissimilated carbon flux to the implemented PHBV-biosynthetic pathway.

2. Materials and Methods

2.1. Bacterial Strains, Plasmids, Editing Template, and Oligonucleotides

Bacterial strains and plasmids used in this study are listed in Table 1, while oligonucleotide sequences are presented in Table S1. Genomic DNA from bacterial cells was isolated using the Blood & Tissue DNA Isolation Kit (Qiagen, Hilden, Germany). Taq DNA polymerase was obtained from New England Biolabs (Ipswich, MA, USA). E. coli HI-Control 10G chemically competent cells (Lucigen, Middleton, WI, USA) were used as the host for plasmid cloning and propagation. The plasmids were extracted using Qiagen Miniprep kit according to the manufacturer’s instructions and confirmed by DNA sequencing conducted by The Centre for Applied Genomics (TCAG) (Toronto, ON, Canada). All plasmids were constructed using Gibson assembly [41] except for the gRNAs which were constructed by ligation. All oligonucleotides were synthesized by Integrated DNA Technologies (IDT) (Coralville, IA, USA). E. coli strains were stored as glycerol stocks at −80 °C.
Standard recombinant DNA technologies were applied for molecular cloning [43]. Activation of the genomic Sbm operon in BWΔldhA to generate E. coli CPC-Sbm was described previously [44].
To construct pDonor1, the gRNA cassette spc.-gRNA.P279T and cas9 were amplified using primer sets P001/P002 and P003/P004, respectively, from a lab-made plasmid for expression of cas9 in E. coli harboring the spc.-gRNA.P279T gRNA cassette and the cas9. Primer sets P005/P006 and P007/P008 were used to amplify the TL and TR sequences, respectively, which are recognized by the integrase to excise the harboring DNA cassette. Primers P009/P010 were used to amplify the backbone from pDonor [22]. Finally, the five fragments were Gibson-assembled to form pDonor1. To construct pDonor2, phaB and bktB were amplified using primer sets P011/P012 and P013/P014, respectively, with the genomic DNA of wild-type Cupriavidus necator ATCC 43291 as the template. The backbone was amplified using primers P015/P016, and the TL and TR sequences were amplified using primer sets P017/P018 and P019/P020 from the pDonor template, respectively. The five fragments were Gibson-assembled to form pDonor2. Similarly, pDonor3 was constructed by amplifying phaC and phaA using primer sets P021/P022 and P023/P024 from the genomic DNA of wild-type C. necator ATCC 43291. The backbone was amplified using primers P015/P016, and the TL and TR sequences were amplified using primer sets P025/P018 and P019/P020 from the pDonor template, respectively. Finally, the five fragments were Gibson-assembled to form pDonor3.
All pEffector-derived plasmids were constructed by assembling three common fragments amplified from the pEffector [22] plasmid using primer sets P026/P027, P028/P029, and P030/P031 and a specific protospacer amplified from a subcloning plasmid using P032/P033. To construct the subcloning plasmids, the protospacer sequences yjcS.P882T, bcsA.P1249NT and intF.P242T were first constructed by annealing the sense and antisense oligonucleotides using P034/P035, P036/P037, and P038/P039, respectively. The annealed oligos were then ligated into BsaI-digested pUC19-derived plasmid in which the ampicillin resistance marker was previously replaced with a spectinomycin cassette.
To construct the pbktB.phaB- Δ bcsA delivery vector, we began by amplifying the bcsA 5′ and 3′ homology lengths (HL-5′ and HL-3′) using primer sets P040/P041 and P042/P043, respectively, from the genomic DNA of E. coli MG1655. The DNA cassette including bktB and phaB was amplified from pDonor2 using primers P044/P045. The fragments were Gibson-assembled with the pTrc99a backbone amplified by primers P046/P047 to construct pbktB.phaB- Δ bcsA. The plasmid was then used as the template to obtain the PCR-amplified editing template using primers P040/P043. Similarly, to construct the pCas9- Δ yjcS delivery vector, we amplified the yjcS HL-5′ and HL-3′ with primer sets P048/P049 and P050/P051, respectively, from the genomic DNA of E. coli MG1655. The gRNA cassette spc.-gRNA.P279T and cas9 were amplified from pDonor1 using primers P052/P053. The fragments were Gibson-assembled with the pTrc99a backbone amplified by primers P054/P055 to construct pCas9- Δ yjcS. The plasmid was then used as the template to obtain the PCR-amplified editing template using primers p048/P051.
The gRNA plasmids harboring gRNA cassettes yjcS.P1032T, bcsA.P1088NT, iclR.P87T, and sdhA.P392T were constructed by amplifying a single fragment using respective forward primers P056, P057, P058, and P059 containing unique protospacers and a common reverse primer, P060. A lab-made plasmid containing PxylA.SphI upstream of a gRNA cassette (described previously [45]) with pBR322-ori was used as the template for pgRNA1 and pgRNA2. Another lab-made plasmid containing PxylA.SphI upstream of a gRNA cassette with pSC101ts-ori was used as the template for pgRNA3 and pgRNA4. Finally, each fragment was self-ligated to form gRNA delivery vectors.
pCas9 was constructed by amplifying the gRNA cassette spc.-gRNA.P279T using primers P061/P062 and amplifying cas9 using primers P063/P064 from pDonor1. The fragments were Gibson-assembled with the two fragments of backbone amplified using primer sets P065/P066 and P067/P068 from pSC101 plasmid.
The genotypes of derived knockout strains were confirmed by colony polymerase chain reaction (PCR) using the appropriate verification primer sets listed in Table S1.

2.2. Competent Cells Preparation and Transformations

To prepare chemically competent cells, bacterial cells on the overnight plate were used to inoculate the prewarmed Luria-Bertani (LB) medium with the initial optical density at 600 nm (OD600) of ~0.1. All experiments for CRISPR-Cas9 coupled with λRed recombineering were conducted at 30 °C and the cultures were induced by 15 mM L-arabinose at OD600 of ~0.3–0.4 to trigger the λRed operon. The cultures for transposon-associated CRISPR-Cas experiments were prepared at 37 °C. Cells were harvested at OD600 of ~0.5 and then washed with ice-cold 15% glycerol three times. Finally, cells were concentrated 230 times and 40 μL of the competent cells were used per transformation. Approximately 250 ng of the donor plasmid or editing template and 100 ng of the Effector or gRNA plasmid were mixed with 40 μL of electrocompetent cells. Mixture of the cells and DNA were electroporated and recovered in 1 mL SOC medium for 1.5 h and then spread on selective LB agar plates and incubated at 30 °C overnight.
For the transposition experiments, prior to restreaking, the bacteria were pooled from the plate by scraping the colonies, resuspending them in LB and appropriately diluting before plating them again. This step is beneficial for obtaining homogeneous colonies since it has been reported that some of the colonies growing on the first plate might be heterogeneous [31].
The derived mutations were confirmed by applying colony PCR toward colonies restreaked from each plate using the appropriate primer sets listed in Table S1. The editing efficiencies of the mutations were reported as the ratio of the successful mutations to the total number of colonies screened for colony PCR (12 colonies per plate).

2.3. Media and Bacterial Cell Cultivation

All media components were purchased from Sigma-Aldrich Co. (St Louis, MO, USA) except glucose, yeast extract, and tryptone which were obtained from BD Diagnostic Systems (Franklin Lakes, NJ, USA). The optical cell density measurements were made by appropriately diluting the samples with 0.15 M saline and using a spectrophotometer (GENESYS™ 40/50 Vis/UV-Vis, Thermo Fisher Scientific Inc., Waltham, MA, USA). When required, the media were supplemented with antibiotics at the following concentrations: 40 μg/mL chloramphenicol and 90 μg/mL spectinomycin. For double selection the spectinomycin concentration was reduced to 80 μg/mL.
E. coli mutants were streaked on LB agar plates and incubated at 30 °C for 16 h. The plates were then used to inoculate 20 mL prewarmed LB broth in 125 mL conical flasks as starter cultures at 30 °C and 280 rpm for 8 h in a rotary shaker (New Brunswick Scientific, NJ, USA). The starter cultures were used to inoculate 215 mL prewarmed super broth (SB) medium (32 g/L tryptone, 20 g/L yeast extract, and 5 g/L NaCl) as seed cultures in 1 L conical flasks. The seed cultures were grown at 30 °C and 280 rpm for 16 h. The cells were then harvested by centrifugation at 5000× g for 10 min and resuspended in 50 mL fresh LB medium which was eventually used to inoculate a 1 L stirred tank bioreactor (CelliGen 115, Eppendorf AG, Hamburg, Germany). The bioreactor cultivations operate at 30 °C and 430 rpm for 24 h and cultivation medium consists of 30 g/L glycerol, 10 g/L yeast extract, 10 mM NaHCO3, 0.4 μM cyanocobalamin (vitamin B12), 0.1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG), 0.23 g/L K2HPO4, 0.51 g/L NH4Cl, 49.8 mg/L MgCl2, 48.1 mg/L K2SO4, 2.78 mg/L FeSO4•7H2O, 0.055 mg/L CaCl2, 2.93 g/L NaCl, 0.72 g/L tricine and 1000th dilution (i.e., 1 mL/L) trace elements (2.86 g/L H3BO3, 1.81 g/L MnCl2•4H2O, 0.222 g/L ZnSO4•7H2O, 0.39 g/L Na2MoO4•2H2O, 79 μg/L CuSO4•5H2O, 49.4 μg/L Co(NO3)2•6H2O). Aerobic conditions were obtained by sparging air into the bulk culture at 1 vvm. The pH of the culture was maintained at 7.0 ± 0.1 using 3 M NH4OH and 3 M H3PO4.

2.4. PHBV Extraction and Analysis

Intracellular polymer was extracted by applying the following procedure. The bacterial cells from the bioreactor cultivations were harvested and centrifuged at 8000× g for 10 min. The pellets were washed twice with distilled water and then lyophilized for 16 h. The dried cell weight (DCW) was measured prior to methanolysis. Methanolysis was carried out at 100 °C for 4 h by resuspending the pellets in 2 mL chloroform and 1 mL PHA solution (i.e., 4 g/L benzoic acid and 15% sulfuric acid in methanol). The biphasic mixtures were then cooled down to room temperature and then were vortexed after adding 1 mL distilled water to them. Finally, after phase separation, the chloroform phase was filtered with 0.45 μm Polytetrafluoroethylene (PTFE) filters, appropriately diluted and then analyzed by Agilent 6890 series GC system (Agilent Technologies, Santa Clara, CA, USA) with a J & W Scientifics DB Wax column (30 m × 0.53 mm, film thickness 1 μM) (Agilent Technologies, Santa Clara, CA, USA). The oven program was set as follows: initial temperature was set at 80 °C for 5 min, then ramped to 230 °C at 7.5 °C/min, and continued to ramp to 260 °C at a faster rate 10 °C/min followed by maintaining that temperature for the analysis. The calibration curves for methanolysis assay were prepared using the standards of methyl 3-hydroxybutyrate and methyl 3-hydroxyvalerate (Sigma-Aldrich Co., St Louis, MO, USA). The PHA content is reported as the ratio of PHA mass measured by GC to the DCW, expressed in weight percent. The 3-HV content is reported as the ratio of 3-HV to the total of 3-HV and 3-HB, expressed in mole percent.

2.5. Statistical Analysis

All experiments in this study were conducted in either triplicate (for genome engineering) or duplicate (for bioreactor cultivations). We used t-test and ANOVA analysis to evaluate our methods and data. Readers are encouraged to check Supplementary Materials for details of each analysis.

3. Results

3.1. Genomic Knock-In Based on CRISPR-Cas9 Coupled with λRed Recombineering

We first explored genomic integration of a DNA cargo (i.e., cas9 as a large-size cargo and bktB:phaB as a small-size cargo) in E. coli using CRISPR-Cas9 coupled with λRed recombineering. To do this, strain ST001 was first derived by inserting the three λRed recombineering genes of exo (α), beta (β), and gam (γ), whose expression was regulated by the arabinose-inducible PBAD promoter, into the endA locus of the E. coli genome. The three recombineering genes were annealed to the FRT-KnR-FRT cassette and the insertion was conducted by Flp-FRT recombination. The FRT-KnR-FRT cassette was then eliminated by transforming the strain with pCP20 [46], a temperature-sensitive plasmid expressing a flippase (Flp) recombinase. Upon Flp-mediated excision of the KnR cassette, a single Flp recognition site was generated. Plasmid pCP20 was then cured by growing cells at 42 °C. Then, ST001 was transformed with pCas9 followed by two rounds of λRed recombineering. In the first round, ST001 harboring pCas9 was co-transformed with pgRNA1 expressing gRNA targeting yjcS and the large-size DNA cargo containing cas9 flanked by 3′ and 5′ homologous arms. Genomic integration of cas9 into the yjcS locus was confirmed by colony PCR using appropriate primer sets (Figure 3A). To prepare for the next round of genome engineering, only pgRNA1 was evicted to form ST002 harboring pCas9. Then, ST002 harboring pCas9 was co-transformed with pgRNA2 expressing gRNA targeting bcsA and the small-size DNA cargo containing bktB:phaB flanked by 3′ and 5′ homologous arms. Finally, after confirming the successful integration of bktB:phaB into the bcsA locus by colony PCR (Figure 3A), both pCas9 and pgRNA2 were evicted to form ST003.
Efficiencies of 91.7% and 22.2% were obtained for genomic integration of the small- and large-size DNA cargo, respectively, with low volumetric CFU for both cases (Figure 3B). Note that both the integration efficiency and volumetric CFU decreased with an increased size of DNA cargo (t-test with p-values of 2.3 × 10−3 and 1.5 × 10−3 respectively), and the integration efficiency appeared to be limited by high numbers of unedited background colonies (also known as escaper colonies) evading the Cas9 break. In addition, low gRNA eviction efficiencies were observed (Figure 3C), potentially due to a high copy number of the plasmid with the pBR322 origin, limiting the applicability of this system for genome engineering.

3.2. Genomic Knock-In Based on Transposon-Associated CRISPR-Cas System

In light of the above technical disadvantages for the CRISPR-Cas9 coupled with λRed recombineering, we further explored transposon-associated CRISPR-Cas system for genomic integration. To do this, ST001 was first used as the host for genomic insertion of cas9 as a large-size cargo into the yjcS locus by co-transforming pDonor1 (which carries cas9) and pEffector1 (which carries the gRNA targeting yjcS). Genomic integration was confirmed by colony PCR using appropriate primer sets (Figure 4A). For the next round of genome engineering, both plasmids were evicted from the resulting engineered strain, generating ST002. Then, the DNA cargo containing bktB:phaB as a small-size cargo was integrated into the bcsA locus of ST002 by co-transforming pDonor2 and pEffector2. Both plasmids were evicted from the resulting engineered strain, generating ST003. Finally, for complete implementation of the PHBV biosynthetic pathway, another DNA cargo containing phaC:phaA as a medium-size cargo was inserted into the intF locus of ST003 by co-transforming pDonor3 and pEffector3. Both plasmids were evicted from the resulting engineered strain, generating ST004.
Efficiencies of 100%, 97.1%, and 88.9% were obtained for genomic integration of a small-, medium-, and large-size cargo, respectively, with high volumetric CFU for all cases (Figure 4B). Although the integration efficiency based on Tn6677 transposition appeared to decrease with an increased DNA cargo size, it was high enough to make the system a feasible toolkit for genome engineering. The ANOVA also confirms that decrease in the integration efficiency is not significant (See Table S2, p-value = 2.7 × 10−1). A similar decreasing trend, though not significant (See Table S3, p-value = 2.3 × 10−1), was observed in volumetric CFU upon increasing the DNA cargo size. In addition, the efficiency of plasmid eviction, particularly for pDonor, also dropped considerably with an increased DNA cargo size (See Table S4, p-value = 2.2 × 10−3) (Figure 4C), potentially limiting the applicability of this system.

3.3. Gene Knockout Based on CRISPR-Cas9 Coupled with λRed Recombineering

Next, we assessed the feasibility of the CRISPR-Cas9 coupled with λRed recombineering system for gene knockout using the iclR and sdhA genes for demonstration. To overcome the low efficiency for eviction of the pgRNA plasmids, the pBR322 origin was replaced with the temperature-sensitive pSC101ts one. To knockout iclR, ST004 was co-transformed with pgRNA3 expressing gRNA targeting iclR and a 60 bp oligonucleotide flanked by 3′ and 5′ homology arms. The oligonucleotide containing six consecutive point mutations was designed to introduce two consecutive stop codons and an AseI restriction site for screening of successful recombination, which was confirmed by colony PCR using appropriate primer sets (Figure 5A). For the next round of gene knockout, pgRNA3 was evicted to form ST005, which was then co-transformed with pgRNA4 expressing gRNA targeting sdhA and the respective oligonucleotide for recombination. After confirming successful knockout by colony PCR, pgRNA4 was evicted from the resulting strain to form ST006.
Efficiencies of 61.1% and 94.4% were obtained for knocking out the iclR and sdhA genes, respectively (Figure 5B). Note that pgRNA plasmids were evicted with 100% efficiency for both rounds of gene knockout (Figure 5C), suggesting the feasibility of this system for genome engineering. Additionally, note that the control experiments using ST007 (without three λRed recombineering genes on the genome) as the host had no genomic integration (data not shown), highlighting the importance of homologous recombination-mediated by the λRed system for efficient gene knockout.

3.4. PHBV Biosynthesis Using Genome-Engineered Strains

PHBV production mainly relies on the availability of the monomer of 3HV-CoA, which is derived from succinyl-CoA in the tricarboxylic acid (TCA) cycle (Figure 2). It was previously shown that, by inactivating the oxidative TCA branch with the sdhA mutation and deregulating the glyoxylate shunt with the iclR mutation, the 3-HV monomeric fraction in the PHBV copolymer could be regulated [47]. A similar demonstration was conducted herein using the three genome-engineered and plasmid-free strains (i.e., ST004, ST005, and ST006) for bioreactor cultivation under aerobic conditions. The results suggest that cell growth was minimally affected by the iclR and sdhA mutations (Figure 6A). Although the DCW was slightly reduced (i.e., 4% and 9% for ST005 and ST006, respectively, compared to ST004), the PHBV content was higher for ST005 (51%) and ST006 (45.9%) compared to ST004 (41.7%). Moreover, the 3-HV monomeric fraction of PHBV copolymer also increased in ST005 (13.2 mol%) and ST006 (17 mol%) compared to ST004 (11 mol%) (t-test with p-values of 4.1 × 10−2 and 2.5 × 10−5 respectively) (Figure 6B). In addition, the 3-HV titer was also increased by inactivating both iclR and sdhA genes, confirming the successful flux channeling into the Sbm pathway (Figure 6C). Additionally, the titers of 3-HB and 3-HV monomers suggest that the PhaC is the limiting factor for PHBV synthesis, an outcome also described previously in E. coli [48]. The results suggest that not only PHBV could be produced by these genome-engineered and plasmid-free strains but also the 3-HV monomeric fraction of PHBV copolymer could be modulated by introducing iclR and sdhA mutations.

4. Discussion

Today, strategies based on CRISPR have been developed for genome engineering of various microorganisms, particularly E. coli. While most of these strategies mainly focus on gene knockout, site-specific insertion of heterologous genes into the genome remains a challenge [49,50] particularly for large-size genes. In this study, we developed an efficient toolkit for both site-specific inactivation of native genes based on a traditional CRISPR-Cas9 coupled with λRed recombineering system (i.e., gene knockout) and site-specific insertion of heterologous genes based on a transposon-associated CRISPR-Cas system (i.e., gene knock-in) in E. coli. With specific engineered strains being developed, the number of plasmids simultaneously used for genome editing was minimized. Additionally, the toolkit showed high editing efficiencies with effective plasmid curing, facilitating multiple rounds for successive genome editing. Using the toolkit, customized engineered strains were developed for heterologous production of PHBV in plasmid-free E. coli.
To evaluate the system of CRISPR-Cas9 coupled with λRed recombineering for gene knock-in, we first constructed a plasmid-free strain ST001 by insertion of a DNA cassette containing the three λRed recombineering genes, i.e., exo (α), beta (β), and gam (γ) whose expression was regulated by the arabinose-inducible PBAD promoter, into E. coli genome. With the expression of the λRed recombineering genes in ST001, homologous recombination was significantly enhanced for genomic insertion of DNA cargos while rejoining the excised genome by CRISPR. Though the λRed recombineering genes might not be critical for conducting CRISPR-mediated gene knockout which is often associated with the use of a small editing template, our results clearly showed that they were required for conducting CRISPR-mediated gene knock-in and the editing efficiency was affected by the size of inserted DNA cargo. It was previously reported that, upon large fragment recombineering via λRed recombineering, the dsDNA cargo was prone to mutations and mismatches, resulting in inefficient DNA recombination [51]. Our findings are also in agreement with a previous report of the size effects of DNA cargo, i.e., ineffective genomic integration of large DNA cargo up to 3 kbp and even failed integration when the size was increased to 3.8 kbp [52]. Similar reduction in integration efficiency was observed by Maresca et al. for DNA insertions greater than 3 kbp while slight decrease in efficiency was reported for gene deletion up to 50 kbp using λRed recombineering [53]. In another study, Jiang et al. attempt to integrate a 4.5 kbp DNA cassette into the yjcS locus yielded the maximum efficiency of 28% [25]. Li et al. also reported significant decrease in integration efficiency from 59% to 14% by increasing the DNA cassette size from 3 kbp to 8 kbp [54]. In addition, the volumetric CFU decreased with an increased size of DNA cargo with the appearance of many unedited escaper colonies. These colonies might have escaped the double-strand break due to homologous recombination between the cleaved and un-cleaved chromosome, deletion of targeted regions, or mutations in the protospacer sequence of the gRNA [55,56], limiting the genome editing efficiency. A critical step for genome engineering is plasmid curing to form the strain for successive genome editing. We observed low plasmid curing efficiency (<10%) in the system of CRISPR-Cas9 coupled with λRed recombineering potentially due to the small size and high copy number of the plasmid. This issue was addressed by changing the plasmid origin to a low-copy one.
Given low editing efficiency upon using CRISPR-Cas9 coupled with λRed recombineering for gene knock-in, we further evaluated the transposon-associated CRISPR-Cas system with a pDonor plasmid carrying the DNA cargo and a pEffector plasmid carrying V. cholerae Tn6677 transposon, type I-F CRISPR-Cas machinery, and a protospacer targeting the desired locus. Upon performing a two-way ANOVA analysis on our results, we concluded that though CRISPR-Cas9 coupled with λRed recombineering might be feasible for integrating DNA cassettes up to ~3 kbp, it was rather inefficient compared to the transposon-associated CRISPR-Cas system (p-value = 3.1 × 10−4) which appeared to be more suitable for integrating larger or pathway-sized DNA cassettes (t-test with p-value = 9.7 × 10−4). The ANOVA analysis also suggest that there is an interaction between the DNA cargo size and the method of integration (See Table S5, p-value = 1.5 × 10−3). The transposon-associated CRISPR system also had much higher volumetric CFU and effective plasmid curing upon genomic integration of large-size DNAs, compared to CRISPR-Cas9 coupled with λRed recombineering. In addition to high editing efficiency and effective plasmid curing, DNA integration via Tn6677 transposons did not require long homologous arms in the DNA cargo, and the integration could occur in a single-step transformation.
Finally, we assessed the feasibility of combining the two systems for multiple rounds of genome engineering, i.e., employing the CRISPR-Cas9 coupled with λRed recombineering system for gene knockout whereas the transposon-associated CRISPR-Cas system for gene knock-in. To facilitate the application, plasmid-free ST002 harboring both the three λRed recombineering genes and cas9 on the genome was used as the base strain such that all genome editing steps could be performed with one-step transformation and plasmid curing. To address the low eviction efficiency for plasmid curing, we used the temperature-sensitive pSC101ts origin to reduce the copy number of the plasmids for genome editing. Furthermore, genomic expression of the cas9 gene will facilitate the eviction of pDonor plasmid when using Tn6677 transposons. For the demonstration using ST002 with an activated Sbm operon on the genome, we first knocked in four pathway genes (i.e., bktB, phaA, phaB, and phaC from C. necator) for heterologous biosynthesis of PHBV. β-ketothiolase which is encoded by phaA and bktB, is involved in two reactions to condensate two acetyl-CoA or an acetyl-CoA with propionyl-CoA to form acetoacetyl-CoA and 3-ketovaleryl-CoA, respectively. Previous studies have shown that bktB exhibits more selectivity towards ketovaleryl-CoA formation, thus more favorable for increasing the 3-HV content of the biopolymer [57]. Acetoacetyl-CoA reductase encoded by phaB reduces the acetoacetyl-CoA and 3-ketovaleryl-CoA to 3-HB-CoA and 3-HV-CoA, respectively. Eventually, polyhydroxyalkanoate synthase encoded by phaC mediates the polymerization of 3-HB-CoA and 3-HV-CoA monomers to form PHBV granules in E. coli cells (Figure 2) [58,59]. Subsequently, we knocked out two native genes (i.e., iclR and sdhA) to direct more dissimilated carbon flux toward the Sbm pathway [60]. Since glycerol has a high degree of reduction and compared to glucose it produces approximately twice the number of reducing equivalents (i.e., NADH) [61], it was used as the carbon source for PHBV production. In addition, glycerol is an abundant byproduct in industry which makes it an inexpensive carbon source [62]. The intermediate plasmid-free ST004, with the PHBV pathway genes being inserted into the genome of ST002, could heterologously produce PHBV at 41.7% of the DCW with 11 mol% of the 3-HV monomeric fraction. With further introduction of two knockouts of ΔiclR and ΔsdhA in ST006, the PHBV content was increased up to 45.9% of the DCW and the 3-HV monomeric fraction to 17 mol% without affecting cell growth. Such metabolic effects for PHBV biosynthesis were similar to those previously reported by Miscevic et al. using engineered E. coli strains with expression plasmids containing PHBV-biosynthetic genes [47]. They have reported slight impacts of iclR and sdhA knockouts on cell growth and total biopolymer content of PHBV (i.e., OD600~35 and DCW~12.5 g/L) in aerobic conditions. They also reported an increase in 3-HV monomeric fraction of the PHBV by knocking these two genes, however, they obtained the 3-HV monomeric fraction of 40.9 mol% which could possibly be due to higher expression levels of the PHBV-biosynthetic genes by expression plasmids.
Overall, our toolkit can be conveniently applied for flexible genome engineering by designing user-specific gRNAs and DNA cargos. Using our base engineered strain, ST002, we could facilitate genome engineering, either gene knockout or knock-in, through a single-step transformation. Moreover, our method relies on the use of curable plasmids which possess low copy number or temperature-sensitive origins. In addition, increasing the expression levels of inserted genes can be achieved by modifying the donor DNA cassette using strong promoters or ribosome binding sites, and even by integrating multiple copies of the donor DNA cassette into different loci in the genome.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/fermentation9010014/s1, Table S1: Oligomers used in this study; Table S2: Statistical analysis data for integration efficiency based on transposon-associated CRISPR-Cas system vs. DNA cargo size; Table S3: Statistical analysis data for volumetric CFU based on transposon-associated CRISPR-Cas system vs. DNA cargo size; Table S4: Statistical analysis data for pDonor eviction efficiency based on transposon-associated CRISPR-Cas system vs. DNA cargo size; Table S5: Statistical analysis data for comparing the efficiency of the integrating methods with increase in the DNA cargo size.

Author Contributions

Conceptualization, B.A. and A.W.; methodology, B.A. and A.W.; validation, B.A.; formal analysis, B.A.; investigation, B.A.; data curation, B.A.; writing—original draft preparation, B.A.; writing—review and editing, M.M.-Y. and C.-H.P.C.; visualization, B.A.; supervision, M.M.-Y. and C.-H.P.C.; funding acquisition, M.M.-Y. and C.-H.P.C. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by Natural Sciences and Engineering Research Council (NSERC) grant number RGPIN-2019-04611.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Not applicable.

Conflicts of Interest

The authors declare no conflict of interest.

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Fermentation 09 00014 g001a 550Fermentation 09 00014 g001b 550
Figure 1. Schematic representation of the two-plasmid system for genomic integration via transposon-associated CRISPR-Cas system. (A) CRISPR-Cas9 coupled with λRed recombineering mechanism: The guide RNA (gRNA) comprised of a protospacer (PS), Cas9-binding hairpin (CBH) and transcriptional terminator (ter), directs the Cas9 to the desired locus by recognizing the PAM sequence and the Cas9 cleaves the DNA through double strand breaks. Exo (lambda exonuclease) generates single-stranded 3′ overhangs and Beta proteins mediate annealing of the homology arms (HA) to the complementary DNA target in the genome. (B) Transposition mechanism: pEffector plasmid harboring the crRNA, VcCascade and transposition machinery. The CRISPR array comprises two repeats (R) and a protospacer (PS) presented in violet. VcCascade consists of Cas6, Cas7 and Cas8 protein subunits presented in green. The transposition machinery comprises TniQ subunit and transposition proteins TnsA, TnsB and TnsC presented in orange. pDonor plasmid harboring a DNA cargo, i.e., bktB:phaB (2.2 kbp), phaC:phaA (3.3 kbp) or cas9 (5.5 kbp), flanked by transposon right- (TR) and left- (TL) end sequences. The crRNA which is principally responsible for the PAM site recognition and R-loop formation, binds to the VcCascade. The TniQ homodimer is bound to the VcCascade to form the VcCascade-crRNA-TniQ complex. To insert the DNA cargo in the desired genomic site, the crRNA directs the VcCascade-crRNA-TniQ complex to the desired locus, TniQ communicates with TnsC, and TnsC mediates the transposition by employing TnsAB to excise the DNA from its pDonor through double strand breaks, recognizing and binding to the TL and TR end sequences of the DNA cassette and integrates the DNA approximately 50 bp downstream of the targeted locus.
Figure 1. Schematic representation of the two-plasmid system for genomic integration via transposon-associated CRISPR-Cas system. (A) CRISPR-Cas9 coupled with λRed recombineering mechanism: The guide RNA (gRNA) comprised of a protospacer (PS), Cas9-binding hairpin (CBH) and transcriptional terminator (ter), directs the Cas9 to the desired locus by recognizing the PAM sequence and the Cas9 cleaves the DNA through double strand breaks. Exo (lambda exonuclease) generates single-stranded 3′ overhangs and Beta proteins mediate annealing of the homology arms (HA) to the complementary DNA target in the genome. (B) Transposition mechanism: pEffector plasmid harboring the crRNA, VcCascade and transposition machinery. The CRISPR array comprises two repeats (R) and a protospacer (PS) presented in violet. VcCascade consists of Cas6, Cas7 and Cas8 protein subunits presented in green. The transposition machinery comprises TniQ subunit and transposition proteins TnsA, TnsB and TnsC presented in orange. pDonor plasmid harboring a DNA cargo, i.e., bktB:phaB (2.2 kbp), phaC:phaA (3.3 kbp) or cas9 (5.5 kbp), flanked by transposon right- (TR) and left- (TL) end sequences. The crRNA which is principally responsible for the PAM site recognition and R-loop formation, binds to the VcCascade. The TniQ homodimer is bound to the VcCascade to form the VcCascade-crRNA-TniQ complex. To insert the DNA cargo in the desired genomic site, the crRNA directs the VcCascade-crRNA-TniQ complex to the desired locus, TniQ communicates with TnsC, and TnsC mediates the transposition by employing TnsAB to excise the DNA from its pDonor through double strand breaks, recognizing and binding to the TL and TR end sequences of the DNA cassette and integrates the DNA approximately 50 bp downstream of the targeted locus.
Fermentation 09 00014 g001aFermentation 09 00014 g001b
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Figure 2. Schematic representation of engineered PHBV pathway in E. coli using glycerol as the carbon source. TCA cycle is presented in black. The reductive branch of the TCA cycle is show with purple arrows. Blue arrows represent the glyoxylate shunt in TCA cycle. The activated Sleeping beauty mutase (Sbm) pathway is presented in orange arrows. PHBV pathway and heterologous enzymes from C. necator are shown in green. Metabolite abbreviations: PHBV, Poly(3-hydroxybutyrate-co-3-hydroxyvalerate); (R)-3-HB-CoA, (R)-3-hydroxybutyryl-CoA; (R)-3-HV-CoA, (R)-3-hydroxyvaleryl-CoA; DHAP, dihydroxyacetone phosphate; PEP, phosphoenolpyruvate. Protein abbreviations: AceA, isocitrate lyase; AceB, malate synthase A; AceK, isocitrate dehydrogenase kinase/phosphatase; IclR, AceBAK operon repressor; LdhA, lactate dehydrogenase A; PckA, phosphoenolpyruvate carboxykinase; PPC, phosphoenolpyruvate carboxylase; PhaA, acetoacetyl-CoA thiolase; PhaB, acetoacetyl-CoA reductase; PhaC, PHA synthase; Sbm, methylmalonyl-CoA mutase; SdhA, succinate dehydrogenase subunit.
Figure 2. Schematic representation of engineered PHBV pathway in E. coli using glycerol as the carbon source. TCA cycle is presented in black. The reductive branch of the TCA cycle is show with purple arrows. Blue arrows represent the glyoxylate shunt in TCA cycle. The activated Sleeping beauty mutase (Sbm) pathway is presented in orange arrows. PHBV pathway and heterologous enzymes from C. necator are shown in green. Metabolite abbreviations: PHBV, Poly(3-hydroxybutyrate-co-3-hydroxyvalerate); (R)-3-HB-CoA, (R)-3-hydroxybutyryl-CoA; (R)-3-HV-CoA, (R)-3-hydroxyvaleryl-CoA; DHAP, dihydroxyacetone phosphate; PEP, phosphoenolpyruvate. Protein abbreviations: AceA, isocitrate lyase; AceB, malate synthase A; AceK, isocitrate dehydrogenase kinase/phosphatase; IclR, AceBAK operon repressor; LdhA, lactate dehydrogenase A; PckA, phosphoenolpyruvate carboxykinase; PPC, phosphoenolpyruvate carboxylase; PhaA, acetoacetyl-CoA thiolase; PhaB, acetoacetyl-CoA reductase; PhaC, PHA synthase; Sbm, methylmalonyl-CoA mutase; SdhA, succinate dehydrogenase subunit.
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Fermentation 09 00014 g003 550
Figure 3. (A) Colony PCR screening of cas9 and bktB:phaB integrations via λRed recombineering (column 2–3). To screen for cas9 integration, primers P069/P070 amplified an 858 bp fragment (column 2). To screen for bktB:phaB integration, primers P071/P072 amplified a 930 bp fragment (column 3). (B) Integration efficiency based on colony PCR (ratio of colonies containing the desired mutation to the total number of colonies screened for colony PCR) and colony count (CFU/mL) of each transformation employing λRed recombineering system to integrate small- and large-size DNA cargos. (C) pgRNA plasmid eviction efficiencies confirmed by spectinomycin sensitivity (ratio of colonies with successful plasmid eviction to total number of colonies screened for antibiotic sensitivity). All values are reported as means ± SD (n = 3).
Figure 3. (A) Colony PCR screening of cas9 and bktB:phaB integrations via λRed recombineering (column 2–3). To screen for cas9 integration, primers P069/P070 amplified an 858 bp fragment (column 2). To screen for bktB:phaB integration, primers P071/P072 amplified a 930 bp fragment (column 3). (B) Integration efficiency based on colony PCR (ratio of colonies containing the desired mutation to the total number of colonies screened for colony PCR) and colony count (CFU/mL) of each transformation employing λRed recombineering system to integrate small- and large-size DNA cargos. (C) pgRNA plasmid eviction efficiencies confirmed by spectinomycin sensitivity (ratio of colonies with successful plasmid eviction to total number of colonies screened for antibiotic sensitivity). All values are reported as means ± SD (n = 3).
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Fermentation 09 00014 g004 550
Figure 4. (A) Colony PCR screening of cas9, bktB:phaB and phaC:phaA integrations via Tn6677 transposons (column 2–4). To screen for cas9 integration, primers P070/P073 amplified a 484 bp fragment (column 2). To screen for bktB:phaB integration, primers P072/P074 amplified a 641 bp fragment (column 3). To screen for phaC:phaA integration, primers P075/P076 amplified a 704 bp fragment (column 4). (B) Integration efficiency (ratio of colonies containing the desired mutation to the total number of colonies screened for colony PCR) and colony count (CFU/mL) of each transformation employing transposon-associated CRISPR-Cas system to integrate small-, medium- and large-size DNA cargos. (C) pDonor and pEffector plasmid eviction efficiencies (ratio of colonies with successful plasmid eviction to total number of colonies screened for antibiotic sensitivity). All values are reported as means ± SD (n = 3).
Figure 4. (A) Colony PCR screening of cas9, bktB:phaB and phaC:phaA integrations via Tn6677 transposons (column 2–4). To screen for cas9 integration, primers P070/P073 amplified a 484 bp fragment (column 2). To screen for bktB:phaB integration, primers P072/P074 amplified a 641 bp fragment (column 3). To screen for phaC:phaA integration, primers P075/P076 amplified a 704 bp fragment (column 4). (B) Integration efficiency (ratio of colonies containing the desired mutation to the total number of colonies screened for colony PCR) and colony count (CFU/mL) of each transformation employing transposon-associated CRISPR-Cas system to integrate small-, medium- and large-size DNA cargos. (C) pDonor and pEffector plasmid eviction efficiencies (ratio of colonies with successful plasmid eviction to total number of colonies screened for antibiotic sensitivity). All values are reported as means ± SD (n = 3).
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Figure 5. (A) Colony PCR screening of iclR and sdhA knockouts via λRed recombineering (column 2–3). To screen for iclR knockout, primers P077/P078 amplified a 790 bp fragment and successful recombination of the editing template generated products of 490 bp and 300 bp upon AseI digestion (columns 2). To screen for sdhA knockout, primers P079/P080 amplified a 1065 bp fragment and successful recombination of the editing template generated products of 779 bp and 286 bp upon AseI digestion (column 3). (B) Gene knockout efficiency based on colony PCR (ratio of colonies containing the desired mutation to the total number of colonies screened for colony PCR) and colony count (CFU/mL) of each transformation employing λRed recombineering system to knockout iclR and sdhA genes. (C) pgRNA plasmid eviction efficiencies confirmed by chloramphenicol sensitivity (ratio of colonies with successful plasmid eviction to total number of colonies screened for antibiotic sensitivity). All values are reported as means ± SD (n = 3).
Figure 5. (A) Colony PCR screening of iclR and sdhA knockouts via λRed recombineering (column 2–3). To screen for iclR knockout, primers P077/P078 amplified a 790 bp fragment and successful recombination of the editing template generated products of 490 bp and 300 bp upon AseI digestion (columns 2). To screen for sdhA knockout, primers P079/P080 amplified a 1065 bp fragment and successful recombination of the editing template generated products of 779 bp and 286 bp upon AseI digestion (column 3). (B) Gene knockout efficiency based on colony PCR (ratio of colonies containing the desired mutation to the total number of colonies screened for colony PCR) and colony count (CFU/mL) of each transformation employing λRed recombineering system to knockout iclR and sdhA genes. (C) pgRNA plasmid eviction efficiencies confirmed by chloramphenicol sensitivity (ratio of colonies with successful plasmid eviction to total number of colonies screened for antibiotic sensitivity). All values are reported as means ± SD (n = 3).
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Figure 6. PHBV biosynthesis of the metabolically manipulated strains (ST004, ST005, ST006) (A) presenting cell optical density (OD600) and dried cell weight (g/L) (B) biopolymer and 3-HV content represented by wt% and mol%, respectively and (C) Metabolite production (g/L). All values are reported as means ± SD (n = 2).
Figure 6. PHBV biosynthesis of the metabolically manipulated strains (ST004, ST005, ST006) (A) presenting cell optical density (OD600) and dried cell weight (g/L) (B) biopolymer and 3-HV content represented by wt% and mol%, respectively and (C) Metabolite production (g/L). All values are reported as means ± SD (n = 2).
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Table
Table 1. E. coli strains and plasmids used in this study.
Table 1. E. coli strains and plasmids used in this study.
NameDescription or Relevant GenotypeSource
E. coli host strains
HI-Control 10GmcrA ∆ (mrr-hsdRMS-mcrBC) endA1 recA1 φ 80dlacZ∆M15 ∆lacX74 araD139 ∆ (ara leu)7697 galU galK rpsL (Strr) nupG λ− tonA Mini-F lacIq1 (Gentr)Lucigen
MG1655K-12; F− λ− rph-1Lab stock
CPC-Sbm BWΔldhA, Ptrc::sbm (i.e., with the FRT-Ptrc cassette replacing the 204-bp up-stream of the Sbm operon)Lab stock
ST001CPC-Sbm endA::(PBAD:: γ : β : α )Lab stock
ST002CPC-Sbm endA::(PBAD:: γ : β : α ) yjcS::(PtetA::spc.-gRNA.P279T-cas9)This study
ST003CPC-Sbm endA::(PBAD:: γ : β : α ) yjcS::(PtetA::spc.-gRNA.P279T-cas9) bcsA::(Pgracmax(T7.RBS)::bktB:phaB)This study
ST004CPC-Sbm endA::(PBAD:: γ : β : α ) yjcS::(PtetA::spc.-gRNA.P279T-cas9) bcsA::(Pgracmax(T7.RBS)::bktB:phaB) intF::(Pgracmax(T7.RBS)::phaC:phaAThis study
ST005ST004 ∆iclRThis study
ST006ST004 ∆iclR ∆sdhAThis study
ST007CPC-Sbm yjcS::(PtetA::spc.-gRNA.P279T-cas9)This study
Plasmids
pDonorDonor plasmid for V. Cholerae transposon system[22]
pEffectorEffector expression for V. Cholerae transposon system[22]
pUC19Subcloning plasmid for generating Effector plasmidsLab stock
pTrc99apBR322 ori, Ampr[42]
pSC101pSC101ts ori, CatrLab stock
pDonor1pBR322 ori, PtetA::spc.-gRNA.P279T-cas9, Spcr This study
pDonor2pBR322 ori, Pgracmax(T7.RBS)::bktB:phaB, SpcrThis study
pDonor3pBR322 ori, Pgracmax(T7.RBS)::phaC:phaA, SpcrThis study
pEffector1pSC101ts ori, PJ23119::yjcS-gRNA.P882T, CatrThis study
pEffector2pSC101ts ori, PJ23119::bcsA-gRNA.P1249NT, CatrThis study
pEffector3pSC101ts ori, PJ23119::intF-gRNA.P242T, CatrThis study
pbktB.phaB- Δ bcsApBR322 ori, Pgracmax(T7.RBS)::bktB:phaB, AmprThis study
pCas9- Δ yjcSpBR322 ori, PtetA::spc.-gRNA.P279T-cas9, AmprThis study
pgRNA1pBR322 ori, PxylA.SphI :: yjcS-gRNA.P1032T, SpcrThis study
pgRNA2pBR322 ori, PxylA.SphI :: bcsA-gRNA.P1088T, SpcrThis study
pgRNA3pSC101ts ori, PxylA.SphI :: iclR-gRNA.P78T, CatrThis study
pgRNA4pSC101ts ori, PxylA.SphI :: sdhA-gRNA.P392T, CatrThis study
pCas9pSC101ts ori, PtetA::spc.-gRNA.P279T-cas9, CatrLab stock
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Arab, B.; Westbrook, A.; Moo-Young, M.; Chou, C.-H.P. A Toolkit for Effective and Successive Genome Engineering of Escherichia coli. Fermentation 2023, 9, 14. https://doi.org/10.3390/fermentation9010014

AMA Style

Arab B, Westbrook A, Moo-Young M, Chou C-HP. A Toolkit for Effective and Successive Genome Engineering of Escherichia coli. Fermentation. 2023; 9(1):14. https://doi.org/10.3390/fermentation9010014

Chicago/Turabian Style

Arab, Bahareh, Adam Westbrook, Murray Moo-Young, and Chih-Hsiung Perry Chou. 2023. "A Toolkit for Effective and Successive Genome Engineering of Escherichia coli" Fermentation 9, no. 1: 14. https://doi.org/10.3390/fermentation9010014

APA Style

Arab, B., Westbrook, A., Moo-Young, M., & Chou, C. -H. P. (2023). A Toolkit for Effective and Successive Genome Engineering of Escherichia coli. Fermentation, 9(1), 14. https://doi.org/10.3390/fermentation9010014

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