Design
Overview
Peroxide repressor PerR has been shown as the key factor in the manipulation of the regulatory mechanism of this defense system against oxidative stress. By now, perR has been identified in various bacteria and is linked to a significant effect in bacterial oxidation sensitivity.
Deletion of a PerR-homologous protein resulted in an obvious increase of tolerance and consumption of intracellular oxygen in obligate anaerobes encountered with aerobic environments. The mutant strains also showed higher resistance to H2O2 and other reactive oxygen species (ROS). This multifunctional protein were proposed to play crucial roles in the oxidative stress defenses in anaerobes [1].
The CRISPR system using for the deletion of perR
Overview
CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)-Cas (CRISPR-associated) system provides sequence-specific immunity against invasion by foreign genetic elements. It carries out its functions by incorporating a small part of the invading DNA sequence, termed as spacer into the CRISPR array [2].
Despite the fact that CRISPR–based technologies have readily been used as powerful and precise platform for genetic manipulations, their applications are generally limited in prokaryotes, especially gram-positive strains like Clostridium, due to the huge size and severe cytotoxicity of the heterogeneous Cas protein effectors.
The wide occurrence of CRISPR-Cas systems in many bacteria holds their great potential for endogenous CRISPR-based prokaryotic engineering techniques. By coupling the DNA targeting activity of endogenous type I CRISPR-Cas systems with the DNA-assisted homologous recombination (HR) mechanism, various genome editing purposes could be achieved. including gene insertion, deletion, and replacement in their natural hosts [3].
It has been reported that the endogenous CRISPR-Cas system within bacteria and archaea can be harnessed for genome editing for the host microorganism [4,5,6]. From the analysis on the genome sequence, we found that C. tyrobutyricum produces a Type I-B CRISPR-Cas system natively. Therefore, we turned to exploit this endogenous CRISPR-Cas system for genome editing in C. tyrobutyricum.
Construction of CRISPR system in Clostridium tyrobutyricum
In our project, we built an endogenous type I-B CRISPR system for the genome editing towards C. tyrobutyricum. In order to knock perR sequence out from the genome of C. tyrobutyricum, 34bp of the perR fragment was chosen to be designed as a speacer sequence, which was then combined together with direct repeat sequences to build the CRSIPR array.
The lactose induced promoter (Plac) were synthesized referring to BBa_K3356007.The plasmid pMTL-Plac was constructed via Gibson assembly to combine the CRISPR box we designed, the lactose induced promoter and the pMTL82151 linearized vector backbone which was obtained by digesting with EcoRI and KpnI endonuclease together. Then, two homologous arms upstream and downstream the perR sequence were constructed by using overlap extension PCR. The recombinant plasmids (pMTL-ΔperR) was constructed by using Seamless Cloning Kit to combine the CRISPR editing box into the BamHI/KpnI-double-digested pMTL-Plac.
The recombined plasmid (pMTL-ΔperR) was attempted to be transformed into C. tyrobutyricum by conjugation with E. coli CA434, subsequently.
Verification of the effects of CRISPR system in C. tyrobutyricum
In the presence of lactose (which we used as the inducer) or its analogues, the expression of the CRISPR array will be induced to be activated, deleting the perR fragment on the genome by the mechanism of endogenous CRISPR.
Gene sequencing technology was used to verify the knockout of perR fragment on the genome.
The oxidative stress tolerance of C. tyrobutyricum ΔperR to aerobic conditions was evaluated using both liquid culture assay and plate spot assay.
The liquid culture assay was monitored by measuring the cell density (OD600) at regularly intervals, and those cultures were placed in both in anaerobic and aerobic conditions controlling the rotate speed of the shaker, at 37℃.
While for plate spot assay, the engineered C. tyrobutyricum were diluted to appropriate concentration and 100 μL diluted culture were cultured on RCM plates anaerobically at 37°C, and the colonies were observed after incubating for 48 h.
Intracellular reactive oxygen species (ROS) assay and intracellular reducing power (NAD+ and NADH) assay were also carried out using different kits to determine the effects of perR deletion on the oxidative-stress tolerance of C. tyrobutyricum.
The system that enables perR to be expressed under regulatory
Overview
It can be seen from the paper that the growth performance of the mutant strains that the constitutive oxygen defence characteristic, which was a result of the deletion of PerR, would come up with high energetic expenses and slow growth under anaerobic conditions. While in the wild strain, using the oxidation sensor PerR as an oxygen-dependent switch allows a temporary response to the immediate problem of oxygen exposure [7].
Our project envisions the construction of perR in a plasmid and assembles downstream of the microaerobic/aerobic induced promoters so that its expression would be regulated by oxygen concentration and reduce the negative impacts of the high-consumption resolutions of toxicity to oxygen on growth.
Verification of performances on vgb promoter
In this part of our project, the key promoter vgb was a microaerobic induced promoter of Vitreoscilla hemoglobin gene. Considering the gene compatibility difference between different host bacteria, we designed the pMTL-Pvgb-bs2 plasmid to determine whether the promoter vgb could work normally in Clostridium tyrobutyricum by detecting the fluorescent expression intensity of fluorescent protein Bs2.
Bs2 fluorescent protein and its application in Clostridium
The green fluorescent protein (GFP) has been one of the most widely used reporter in bioprocess monitoring of gene expression. However, they are not functional under anaerobic conditions, and thus cannot be employed as reporters in Clostridium.
A series of flavin mononucleotide (FMN)-based fluorescent proteins (FbFPs) have been reported, which could exhibit strong signals in the absence of O2. FbFPs have been successfully used as a fluorescent label in anaerobic or facultative anaerobic bacteria, including several species of Clostridium for monitoring of protein expression, evaluation of promoter strength, and for proof-of-concept demonstration of transcriptional repression, etc.
The promoter strength and protein expression level of a specific gene in Clostridium could be measured by monitoring the fluorescence intensity, as also reported previously [8]. Therefore, we used the Bs2 fluorescent protein as a reporter to verify the expression performance of the vgb promoter, and detected the intensity of the expression of fluorescence under vgb promoter in both aerobic and microaerobic conditions, respectively.
Fluorescence performance test
The engineered bacteria bearing pMTL-Pvgb-bs2, pMTL-Pthl-bs2 and pMTL82151 plasmids were precultured, inoculated into LB culture medium with different dissolved oxygen, and cultured to OD600 ~0.7 and ~1.2, respectively. The subsequent operation was as recorded by protocol, measuring the fluorescence intensity, collating and the data was analyzed.
The vgb promoter performed a low expression under aerobic conditions, while under microaerobic conditions, it expressed 10.2 folds increased on fluorescence intensity comparing with the constituent promoter, which is exactly as expected.
Using vgb promoter for the regulated expression of perR
Since the vgb promoter exhibited excellent performances when expressing Bs2, which was a low expression under aerobic conditions and extremely high expression under microaerobic conditions, we planned to use Pvgb as the promoter that regulates the expression of perR.
Rational designs of promoter vgb
Promoter engineering: rational design strategies on the improvement of Pvgb.
Since the expectation of our project for the expression of PerR protein is to be highly expressed under anaerobic conditions while lowly under aerobic or microaerobic conditions, we chose microaerobic-induced promoter vgb to regulate the perR sequence.
However, after reviewing the literature, it was found that although this promoter performed prominently among several microaerobic-induced promoters, there were still some problems, such as the fact that it could been induced even at 40% dissolved oxygen(DO) level, and the inducing ratio was quite low [9,10].
Therefore, we tried to improve vgb promoter through promoter engineering strategies [11,12].
Method 1-Doubling promoter vgb enhances the expression level of the promoter
We decided to insert a repeated Pvgb fragment into the upstream of Pvgb, hoping that the expression of Bs2 fluorescent protein could be improved.
Method 2-Inserting exogenous 5'-UTR sequences downstream of the promoter vgb significantly enhances the expression effects of the promoter.
In iGEM's official part registry catalog, we retrieved a translation enhanceing 5-UTR fragment(Part:BBa_K1758100) designed by team Bielefeld-CeBiTec in 2015, and we decided to insert this fragment downstream of the vgb promoter in the pre-constructed plasmid pMTL-vgb-bs2, hoping that the expression of the Bs2 fluorescent protein would be improved.
Method 3-Adjusting the distance between FNR binding site and the -35 region of promoter vgb fine tunes the inhibitory effect of oxygen on the promoter.
To adjust the distance of FNR binding site from the -35 region, we used site-directed mutagenesis. Megaprimer mutation technique was used to separate the FNR binding site from the -35 region by, changing the distance between the FNR binding site to 3bp and 7bp from -35 region, while changing the sequence from the second half of the FNR binding site and the -35 region to more conservative sequences [2,] with higher expressive effects, and reducing the interval speacer between-35 and-10 region to 17bp.
How will we test it?
By controlling aerobic and micro-aerobic culture conditions, we incubated E. coli bearing recombinant plasmid Pvgb-5’-UTR-bs2 together with Pvgb-bs2 as control, under those two conditions respectively, and sampled the culture solutions after logarithmic stages to detect fluorescence intensity after relevant treatment documented in our protocol.
The promoter engineering towards PthlA
As we hoped that the expression of perR genes could be regulated to be a high level under aerobic conditions while stay low under anaerobic conditions.
Therefore, the newfangled idea came into being that the constitutive promoter thlA from C. acetobutylicum might be engineered through a promoter engineering strategy so that it could repress the expression of perR in an aerobic condition as we wish. Through the search of the paper, we decided to regulate thlA promoter by FNR-based oxygen-related biosensors [13].
By inserting the FNR binding site (TTGATttacATCAA)upstream the -35 region of the thlA promoter, we designed the plasmid pMTL-PthlAF7-bs2. After consulting experts of related fields, we adjusted the distance between the FNR binding site and promoter -35 region from 7 bp to 3 bp for a better expecting expression effect [13], and designed a new plasmid pMTL-PthlAF3-bs2.
We hope to verify our experimental design of the improvement on the expression effects of these promoters through the intensity of BS2 fluorescent protein under their expressions [8].
References
[1] Zhang, L., Nie, X., Ravcheev, D.A., Rodionov, D.A., Sheng, J., Gu, Y., et al. (2014) Redox-responsive repressor Rex modulates alcohol production and oxidative stress tolerance in clostridium acetobutylicum. J Bacteriol 196: 3949–3963.
[2] Veena Devi et al. CRISPR‑Cas systems: role in cellular processes beyond adaptive immunity. Folia Microbiological （2022）
[3] Zheng Y, Li J, Wang B, Han J, Hao Y, Wang S, Ma X, Yang S, Ma L, Yi L and Peng W (2020) Endogenous Type I CRISPR-Cas: From Foreign DNA Defense to Prokaryotic Engineering. Front. Bioeng. Biotechnol. 8:62.
[4] Li. YJ, et al. Harnessing Type I and Type III CRISPR-Cas systems for genome editing. Nucleic Acids Research, 2016, Vol. 44, No. 4 e34
[5] Yanli Zheng et al. Endogenous Type I CRISPR-Cas: From Foreign DNA Defense to Prokaryotic Engineering. Frontiers in Bioengineering and Biotechnology March 2020 Volume 8 Article 62
[6] Anna Maikova et al. Using an Endogenous CRISPR-Cas System for Genome Editing in the Human Pathogen Clostridium difficile. Appl. Environ. Microbio October 2019 Volume 85 Issue 20
[7] Falk Hillmann et.al PerR acts as a switch for oxygen tolerance in the strict anaerobe Clostridium acetobutylicum. Molecular Microbiology (2008) 68(4), 848–860
[8] Chi Cheng, et al. Development of an in vivo fluorescence based gene expression reporter system for Clostridium tyrobutyricum. Journal of Biotechnology 305 (2019) 18–22
[9] Jintae Lee, Moo Hwan Cho, and Jongwon Lee, Characterization of an Oxygen-Dependent Inducible Promoter System, the nar Promoter, and Escherichia coli with an Inactivated nar Operon. Biotechnology and Bioengineering, Vol. 52, Pp. 572-578 (1996)
[10] Alvaro R. Lara, et al. Characterization of Endogenous and Reduced Promoters for Oxygen-Limited Processes Using Escherichia coli. ACS Synth. Biol. 2017, 6, 344−356
[11] Ning Xu, Liang Wei, Jun Liu, Recent advances in the applications of promoter engineering for the optimization of metabolite biosynthesis [J]. World Journal of Microbiology and Biotechnology (2019) 35:33
[12] Li-Qun Jin, et al. Promoter engineering strategies for the overproduction of valuable metabolites in microbes. APPLIED MICROBIOLOGY AND BIOTECHNOLOGY 2019
[13] Tingting Hao, Guohui Li, Shenghu Zhou, and Yu Deng. Engineering the Reductive TCA Pathway to Dynamically Regulate the Biosynthesis of Adipic Acid in Escherichia coli. ACS Synth. Biol. 2021, 10, 632−639