Method for regulating and controlling electron current of dissimilatory metal reducing bacteria
阅读说明：本技术 调控异化金属还原菌电子流的方法 (Method for regulating and controlling electron current of dissimilatory metal reducing bacteria ) 是由 俞汉青 李�杰 汤强 刘东风 于 2020-12-30 设计创作，主要内容包括：一种基于CRISPR-ddAsCpf1调控异化金属还原菌电子流的方法,包括如下步骤：(A)初步选择与能量代谢有关的系列基因；(B)构建靶向所述系列基因的crRNA库；(C)将含有crRNA库的质粒和编码ddAsCpf1的质粒转入异化金属还原菌内,并筛选能够调控异化金属还原菌的电子流的有效基因；(D)通过调控所述有效基因的表达来调控异化金属还原菌电子流。(A method for regulating and controlling electron current of dissimilatory metal-reducing bacteria based on CRISPR-ddAsCpf1 comprises the following steps: (A) preliminarily selecting a series of genes related to energy metabolism; (B) constructing a crRNA library targeting the series of genes; (C) transferring plasmids containing a crRNA library and plasmids coding ddAsCpf1 into the dissimilatory metal-reducing bacteria, and screening effective genes capable of regulating and controlling the electron current of the dissimilatory metal-reducing bacteria; (D) the electron current of the dissimilatory metal reducing bacteria is regulated and controlled by regulating and controlling the expression of the effective genes.)
1. a method for regulating and controlling electron current of dissimilatory metal-reducing bacteria based on CRISPR-ddAsCpf1 comprises the following steps:
(A) preliminarily selecting a series of genes related to energy metabolism;
(B) constructing a crRNA library targeting the series of genes;
(C) transferring plasmids containing a crRNA library and plasmids coding ddAsCpf1 into the dissimilatory metal-reducing bacteria, and screening effective genes capable of regulating and controlling the electron current of the dissimilatory metal-reducing bacteria;
(D) the electron current of the dissimilatory metal reducing bacteria is regulated and controlled by regulating and controlling the expression of the effective genes.
2. The method according to claim 1, wherein the series of genes involved in energy metabolism is a cytochrome gene.
3. The method of claim 1, wherein the plasmid containing the crRNA library is a plasmid consisting of a constitutive promoter PCNpECR plasmids expressing crRNA.
4. The method of claim 1, wherein the plasmid encoding ddAsCpf1 is IPTG-inducible PlacPromoter expression pEAC01 plasmid of ddAsCpf 1.
5. The method of claim 1, wherein the dissimilatory metal-reducing bacterium is Shewanella oneidensis MR-1 (Shewanella oneidensis MR-1).
6. The method of claim 1, wherein WO is utilized3And the nanoprobe represents the change of the extracellular electron transfer capability of the dissimilatory metal reducing bacteria.
7. A method for improving the ability of bacteria to reduce pollutants, comprising screening effective genes capable of regulating the electron flow of a dissimilatory metal-reducing bacterium by the method of any one of claims 1 to 6, and improving the extracellular electron transfer ability of the dissimilatory metal-reducing bacterium by inhibiting the expression of the effective genes.
8. The method of claim 7, wherein the dissimilatory metal-reducing bacterium is Shewanella oneidensis MR-1 (Shewanella oneidensis MR-1).
9. The process of claim 8 wherein the contaminant is hexavalent chromium and the effective gene is selected from one or more of SO0717, napB, dmsE, SO2930 and nrfA.
Technical Field
The invention belongs to the field of genetic engineering, and relates to a method for regulating and controlling the electron flow of dissimilatory metal reducing bacteria by inhibiting an electron flow competition path.
Background
The dissimilatory metal reducing bacteria have a special extracellular electron transfer mode for completing the respiratory metabolism process of cells by utilizing a plurality of electron acceptors in an anaerobic environment[1-3]. The bacteria also have important functions in the aspects of pollutant degradation, energy recovery, chemical production, environmental remediation and the like due to the characteristics. Various dissimilatory metal-reducing bacteria, such as Shewanella spp, Geobacter spp, Pseudomonas spp, Listeria spp, have been screened for in different habitats and characterized for their corresponding extracellular electron transport pathways. Shewanella oneidensis MR-1 has been widely used in scientific research as a strain of dissimilatory metal reduction model. Shewanella oneidensis MR-1 has a complex extracellular electron transport network for transporting electrons produced by intracellular metabolism to extracellular electron receptors. The bacteria firstly gather electrons generated by the oxidative decomposition of L-lactic acid in an intracellular manner to an NADH pool, and then transmit the electrons to a plurality of redox terminal enzymes on an inner membrane and a periplasm and a porin-cytochrome complex (OmcA-mtrCAB) on the outer membrane through a quinone pool and finally transmit the electrons to an extracellular electron acceptor in a direct and indirect manner.
Since the characteristic of low natural extracellular electron transfer rate of wild-type dissimilatory metal-reducing bacteria seriously restricts the practical application process of the bacteria, scientific research on how to improve the extracellular electron transfer rate is paid considerable attention and attention. For example, methods such as carbon nano-tube, porous nitrogen deposition carbon cloth, electroactive reduced graphene-oxide hybrid biological membrane and the like are used for establishing a better conductive bacteria-electrode interface, so that the extracellular electron transfer capability of Shewanella oneidensis MR-1 can be enhanced. Recently, synthetic biological methods and techniques have been applied to enhance the ability of transferring electrons from the outside of cells and have shown great potential. For example, modular construction of NADH synthesis pathway increases intracellular NADH pool capacity, overexpression of intracellular riboflavin synthesis gene increases riboflavin synthesis level, and overexpression of MR-1 intracellular C-di-GMP synthesis gene ydeH and CAMP synthesis gene cyaC both achieve the effect of obviously enhancing extracellular electron transfer rate of Shewanella oneidensis MR-1. However, there is still a lack of robust synthetic biological methods for enhancing the ability of bacteria to transfer electrons extracellularly.
The development of gene editing technology based on CRISPR system is continuous, thus providing new possibility for engineering bacteria. The gene silencing technology based on the inactivated dCas9 can not cause DNA double strand break when being combined with a target DNA sequence in a genome in a cell, thereby avoiding the problem of low chromosome adhesion rate, and the technology is widely applied to various prokaryotic cells at present. Recently, class 2 CRISPR-Cas RNA-guided endonuclease Cpf1 was demonstrated to be another highly efficient gene editing tool. Three Cpf1 proteins have been identified as being effective, including AsCpf1 from Acidonococcus spp. BV3L6, Fncpf1 from Franciella novicida and 2YTCpf1 from Lachnospiraceae. CRISPR-Cpf1 has a number of advantages over spCas9 as a gene editing tool. First, only a single crRNA is required to direct Cpf1 to target the target sequence without the need to transactivate the crRNA complex; second, Cpf1 has rnase activity of crRNA precursors; third, Cpf1 recognizes thymine T-rich PAM sequences, thereby expanding its DNA targeting range. These features motivate our use of the CRISPR-Cpf1 tool to attempt to modulate the extracellular electron transport process of dissimilatory metal-reducing bacteria.
Many energy metabolism-related proteins participate in the process of cellular energy distribution by transferring electron flow to different terminal electron acceptors. The shunting and divergence of electrons in the transfer process leads to the reduction of the electron equivalent finally transferred to the extracellular space, thereby reducing the practical application value of dissimilatory metal-reducing bacteria.
Therefore, it is necessary to develop a method for simply and rapidly regulating and controlling the electron current of the dissimilatory metal-reducing bacteria, and the application of the method to the engineering transformation of the dissimilatory metal-reducing bacteria is helpful for the application of the bacteria in the fields of environmental energy and the like.
Disclosure of Invention
The invention aims to provide a method for regulating and controlling the electron current of a dissimilatory metal-reducing bacterium based on CRISPR-ddAsCpf 1.
The embodiment of the invention provides a double-plasmid CRISPR-ddAsCpf1 system for specific screening and inhibitionA cytochrome gene of dissimilatory metal-reducing bacterium Shewanella oneidensis MR-1 for enhancing the extracellular electron transfer capability of the bacterium. One of the plasmids is IPTG-induced PlacPromoter expression pEAC01 plasmid of ddAsCpf1, another plasmid is composed of constitutive promoter PCNpECR plasmids expressing crRNA. In the examples of the present invention, the gfp-lacZ gene cluster used was located within the genome of the MR-1 bacterium. The CRISPR-ddAsCpf1 inhibitory strength was judged by targeting CRISPR-ddAsCpf1 to different binding sites and testing the bacterial GFP fluorescence signal.
The invention provides a method for regulating and controlling electron current of dissimilatory metal-reducing bacteria based on CRISPR-ddAsCpf1, which comprises the following steps:
(A) a preliminarily selected series of genes related to energy metabolism;
(B) constructing a crRNA library targeting the series of genes;
(C) transferring plasmids containing a crRNA library and plasmids coding ddAsCpf1 into the dissimilatory metal-reducing bacteria, and screening effective genes capable of regulating and controlling the electron current of the dissimilatory metal-reducing bacteria;
(D) the electron current of the dissimilatory metal reducing bacteria is regulated and controlled by regulating and controlling the expression of the effective genes.
In some embodiments, the set of genes associated with energy metabolism is a cytochrome gene.
In some embodiments, the plasmid containing the crRNA library is composed of a constitutive promoter PCNpECR plasmids expressing crRNA.
In some embodiments, the plasmid encoding ddAsCpf1 is P inducible by IPTGlacPromoter expression pEAC01 plasmid of ddAsCpf 1.
In some embodiments, the dissimilatory metal-reducing bacterium is a Shewanella oneidensis MR-1 bacterium (Shewanella oneidensis MR-1).
In some embodiments, WO is utilized3And the nanoprobe represents the change of the extracellular electron transfer capability of the dissimilatory metal reducing bacteria.
The invention also provides a method for improving the capability of bacteria to reduce pollutants, which comprises the steps of screening effective genes capable of regulating and controlling the electron current of the dissimilatory metal reducing bacteria by using the method, and improving the extracellular electron transfer capability of the dissimilatory metal reducing bacteria by inhibiting the expression of the effective genes.
In some embodiments, the dissimilatory metal-reducing bacterium is a Shewanella oneidensis MR-1 bacterium (Shewanella oneidensis MR-1).
In some embodiments, the contaminant is hexavalent chromium and the effective gene is selected from one or more of SO0717, napB, dmsE, SO2930, and nrfA.
The invention provides a way for effectively increasing the extracellular electron transfer capacity of bacteria and further promoting bacteria to reduce pollutants, and lays a foundation for designing dissimilatory metal reducing bacteria to carry out more effective environmental remediation applications (such as treating dye-rich wastewater and remedying heavy metal polluted soil and underground water) by using a synthetic biological tool.
Drawings
The drawings are only for purposes of illustrating and explaining the present invention and are not to be construed as limiting the scope of the present invention.
FIG. 1 is a schematic diagram of a dual-plasmid CRISPR-ddAsCpf1 system for inhibiting gfp-lacZ gene cluster in the genome of MR-1 bacteria and effect verification. Schematic diagram of gene elimination for synthesis of controlled vectors. CmR: chloramphenicol; tet (Tet)R: a tetracycline; a terminator: T1T 2; regulatory protein lacI; IPTG inducible promoter PLac(ii) a Constitutive promoter PCN。
FIG. 2 shows the CRISPR-ddAsCpf1 target optimization and effect verification.
FIG. 3 shows CRISPR-ddAsCpf1 specific inhibition and screening of genes for enhanced extracellular electron transport ability.
FIG. 4 shows the CRISPR-ddAsCpf1 screening MR-1 Cr (VI) reduction capability enhancing target gene.
Detailed Description
In order that the objects, technical solutions and advantages of the present invention will become more apparent, the present invention will be further described in detail with reference to the accompanying drawings in conjunction with the following specific embodiments.
The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.
Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
Example 1 construction of the Dual-plasmid CRISPR-ddAsCpf1 System and verification of the Effect of enhancing extracellular Electron transfer
Materials and methods
1. Strain, plasmid and culture method
All strains and plasmids used in this example are listed in Table 1. Among them, E.coli NEB 10. beta. was used as a cloning strain and for phenotypic verification of the genetic circuit. Coli WM3604 was used for conjugation transfer. All E.coli and Shewanella were cultured in 2YT medium and E.coli WM3604 was cultured supplemented with DAP at a final concentration of 50. mu.M. Escherichia coli was cultured at 37 ℃ and Shewanella was cultured at 30 ℃. The composition of the 2YT medium was: 16g/L of peptone, 10g/L of yeast powder and 5g/L of NaCl. 15g/L agar was added to the solid medium.
The above strains are added with antibiotics according to the requirements: 30. mu.g/mL chloramphenicol (E.coli), 10. mu.g/mL chloramphenicol (Shewanella), 10. mu.g/mL tetracycline (E.coli), 5. mu.g/mL tetracycline (Shewanella). IPTG was added as an inducer to the medium at the concentrations noted herein.
The strain was cultured in an incubator by standing or in a shaker at 200 rpm. The bacterial growth was measured by using a BioSpec-1601 ultraviolet spectrophotometer (Shimadzu Co., Japan) to measure the light absorption value (OD600) at a wavelength of 600 nm.
TABLE 1 strains and plasmids used in the examples of the invention
2. Sequence analysis
The complete sequence data of the genome of Shewanella oneidensis MR-1 was obtained from GenBank (accession No.: NC-004347.2).
3. DNA manipulation
The Shewanella whole genome is extracted by E.Z.N.A whole genome DNA extraction kit (OMEGA, Beijing, China). Plasmids were extracted by e.z.n.a plasmid miniprep kit (OMEGA, beijing, china). All DNA fragments used for plasmid construction were purified by gel cutting using e.z.n.a gel recovery kit (OMEGA, beijing, china). The enzyme used for PCR amplification, the enzyme used for preparation of Gibson reaction solution, and the restriction enzyme were purchased from NEB corporation (NEB, Beijing, China). Q5 High-Fidelity DNA polymerase was used to amplify the fragments used for plasmid construction, and OneTaq 2X Master Mix was used for PCR validation. The above experimental procedures were all used according to the product specifications.
4. DNA sequencing
The constructed plasmid, and the PCR-verified amplified product, are sequenced and verified to confirm that it is the target vector or PCR product. Sequencing was performed by Genewiz (Beijing, China) or Rui Bo (Beijing, China).
5. Preparation of E.coli electrotransformation competence
1) Coli growth of 2YT plate from single colony, transferred to containing 100mL 2YT liquid medium in the triangle bottle, in the shaking table 150rpm culture to OD600When the temperature reached 0.6, the flask was taken out and ice-cooled for 25min, during which time the flask was gently shaken every 5 min.
2) Collecting bacteria, subpackaging the bacteria liquid into 50mL centrifuge tubes on a clean bench, balancing, and centrifuging at 4000rpm for 15min at 4 ℃.
3) Removing supernatant, suspending the cells with 10% glycerol precooled by an ice bath, gently blowing the suspended cells with a pipette gun, completely suspending and dispersing the precipitated cells, and centrifuging to collect the bacteria.
4) Repeating the step 3) once.
5) Centrifuge, discard the supernatant, resuspend the cells with 1mL of 10% glycerol. Competent cells were dispensed into pre-cooled EP tubes at 100. mu.L per tube and frozen at-80 ℃ until use.
6. DNA ligation
The method for constructing the plasmid is mainly by means of Gibson ligation.
1) 6mL of 5X ISO Buffer was prepared, with the following composition:
3mL 1M Tris-HCL(pH 7.5)
+150μL 2M MgCl2
+240μL 2M 100mM dNTP mix(25mM each：dGTP，dCTP，dATP，dTTP)
+300μL 1M DTT
+1.5g PEG-8000
+ 300. mu.L of 100mM NAD
+ddH2O to 6mL, and storing in 160 μ L at-20 deg.C for use.
2) 600 μ L of Gibson ligation MIX was prepared with the following composition:
160μL 5X ISO Buffer
+0.32μL 10U/μL T5 exonuclease
+10μL 2U/μL Q5 DNA polymerase
+80μL 40U/μL Taq DNA ligase
+ddH2o to 1.2mL, and the mixture was dispensed into 15. mu.L PCR tubes and stored at-20 ℃ until use.
3) When in use, a 15. mu.L Gibson is taken out to be connected with MIX, and the mixture is placed on ice for dissolving and standby.
4) Plasmid-constructed fragments were prepared and DNA concentration (ng/. mu.L) was determined using NanoDrop (thermo).
5) Mixing 100ng of plasmid vector backbone with equimolar amounts of other fragments, pipetting many times with a pipette gun, mixing well with Gibson's ligation MIX to a final volume of 20. mu.L and ddH for the deficient fraction2And (4) complementing O.
6) The temperature is kept at 50 ℃ for at least 60 minutes.
7) The MIX reaction was removed, and 1-5. mu.L of the reaction solution was pipetted into E.coli competence and electroporated.
7. Step of electrotransformation
1) The plasmid or Gibson reaction solution was mixed with competence, transferred to a 2mm cuvette and allowed to stand on ice for 5 minutes.
2) The electric rotator (Bio-rad) was set to 2.5KV for electric shock, and then 1mL of 2YT medium was immediately added, and the mixture was inverted and mixed.
3) Cells suspended in 2YT medium were transferred to 1mL EP tubes, recovered for 1 hour, plated on correspondingly resistant plates, and placed in an incubator for inverted culture.
8. Plasmid construction
The primers used for plasmid construction are listed in Table 2.
TABLE 2 primers used in the examples of the present invention
The plasmid was constructed as follows: adding LacI-PLac-GFP-T1T2The fragment is selected from pEC01K-PLACAmplified from plasmid and inserted into PMT plasmid skeleton to construct pMT-PLac-GFP. The ddAsCpf1 gene fragment was amplified from pXX55-ddAsCpf145 and used to replace pMT-PLacThe GFP gene of the GFP plasmid was constructed as pEAC01 plasmid. The plasmid pECR-DR was constructed by sequential assembly of the PCN promoter, the forward repeat (DR sequence), and the T1T2 double terminator within the plasmid pCN 00146. Construction of guide RNA library (crRNA) plasmids different crRNA sequences were introduced by reverse PCR amplification of the pECR-DR plasmid. 9. Measurement of bacterial growth and fluorescence
Overnight culture of Shewanella oneidensis GZ bacteria containing the CRISPR-ddAsCpf1 system was transferred to fresh 2YT medium and IPTG inducer added at the corresponding concentration. 200. mu.L of the suspension was put into a culture well of a 96-well plate (Corning Costar, cat. #3603), placed in a microplate reader (BioTek, SynergyH4), continuously cultured at 30 ℃ and measured for growth (absorption light 600nm) and GFP green fluorescence (excitation light wavelength 488 nm; detection wavelength 520 nm).
10、WO3Color development experiment for characterizing the transfer rate of extracellular electrons
WO3The nanoprobes were used to characterize the change in extracellular electron transfer capability of Shewanella bacteria. WO for characterizing the extracellular electron transport capability of bacteria3The nanoprobe is according to the literature[4]And (4) synthesizing. Bacteria were cultured in 2YT medium at 30 ℃ for 1 hour before the addition of IPTG at a final concentration of 5mM to induce expression of ddAsCpf 1. After 24 hours of culture, the cells were collected by centrifugation and washed once with sodium lactate mineral salt medium. Then resuspended to OD in an anaerobic glove box (DG250, Don Whitley Scientific Co., UK) using deoxygenated lactate mineral salt medium6000.2 of bacterial liquid. Transferring the bacterial liquid to a 96-well plate, and adding WO3And (4) a probe is used, and paraffin oil is added to seal the liquid level at the uppermost layer. Testing WO with a scanner after reacting for 2h in a dark place3Blue intensity after probe development. The deeper the blue color indicates the stronger the extracellular electron transport ability of the bacterium.
Second, experimental results
1. Establishing a two-plasmid encoded CRISPR-ddAsCpf1 system in Shewanella oneidensis MR-1 cells
The two-plasmid encoded CRISPR-ddAsCpf1 system was designed and applied within sheva bacterial cells, where the ddAsCpf1 protein and crRNA were encoded by different plasmids, respectively (fig. 1A). This design allows the construction of gene-targeting crRNA plasmids quickly and easily. The ddAsCpf1 gene was controlled by an IPTG-inducible promoter on pEAC01 plasmid carrying a tetracycline resistance marker. The inducible promoter is used to control the expression of the ddAsCpf1 gene in order to reduce metabolic burden and growth inhibition. Efficient gene expression can be achieved by adding IPTG (0-10 mM). The guide RNA (crRNA) is encoded by pECR series plasmids carrying a chloramphenicol resistance gene. Since studies have shown that increasing the expression level of sgrnas helps to increase genome editing efficiency, we designed crrnas to be expressed from a strong constitutive PCN promoter as well.
Thereafter, the GFP-lacZ tandem reporter gene that had been integrated into the genome of Shewanella was used as a targeting gene for the CRISPR-ddAsCpf1 system to examine the feasibility of gene silencing by the ddAsCpf1-crRNA system that had been constructed (FIG. 1B). The induced ddAsCpf1 protein forms a ddAsCpf1-crRNA complex with constitutively expressed crRNA, thereby physically blocking RNA polymerase-catalyzed transcription reaction. The crRNA fragment contains a 23nt base-pairing region directly behind the DR sequence (FIG. 1C). In constructing the crRNA plasmid, the crRNA target sequence can be easily introduced by reverse amplification PCR technique using primers containing the target-specific 23-nt base-pairing region as an overhang. Then, crrnas were designed to target T1 targets just downstream of the Ribosome Binding Site (RBS) on the gfp reporter template strand, and the corresponding crRNA plasmid pECR-T1 was constructed. It was transformed with ddAsCpf1 encoding plasmid pEAC01 to elicit a gene silencing response. Results compared to control, the bacteria in which T1 target was bound by CRISPR-ddAsCpf1 were observed under microscope for complete disappearance of GFP fluorescence (fig. 1D), indicating that the CRISPR-ddAsCpf1 system achieves efficient gene silencing intracellularly in shewanella bacteria.
2. Characterization of influence of CRISPR-ddAsCpf1 binding to different targets on gene silencing effect
To facilitate the use of CRISPR-ddAsCpf1 in rapidly targeting gene sites, we further investigated the effect of different crRNA targeting sites on gene silencing effects. First we designed and constructed three crRNA plasmids targeting NT1, P1 and NP1, respectively (fig. 2A). Where P1 is located on the DNA template strand, it is identical to T1. NP1 and NT1 are on the non-template strand. P1 and NP1 were located downstream of the PL promoter, while T1 and NT1 were located downstream of RBS 1. CRISPR-ddAsCpf1 was induced to target these sites with 5mM IPTG and the effect of binding targets on the gene silencing effect of CRISPR-ddAsCpf1 was assessed by changes in bacterial GFP fluorescence intensity. As a result, it was found that both crRNA-T1 and crRNA-NT1 resulted in complete quenching of bacterial GFP fluorescence, achieving approximately 100% intensity inhibition of GFP gene expression levels (FIG. 2B). While crRNA-P1 and crRNA-NP1 resulted in 60.42% and 63.87% decrease in bacterial GFP fluorescence at 18h, respectively. Taken together, these results show that: binding of ddAsCpf1 to the target on either template or non-template DNA has limited effect on the gene silencing effect. Selecting a binding target downstream of the RBS may inhibit gene expression more effectively than a target downstream of the promoter.
3. Regulation of Shewanella bacterial electron flow by CRISPR-ddAsCpf1 system
The established CRISPR-ddAsCpf1 system was next used to re-alter the electron flux in S.oneidensis MR-1 to increase its extracellular electron transfer rate. We selected ten representative cytochrome genes involved in the energy metabolism of Salmonella MR-1 for testing (FIG. 3A) and established the corresponding crRNA pools to target these genes located in the genome (Table 3).
Table 3: selected 10 cytochrome Gene messages in Shewanella cells
After further validation of the CRISPR-ddAsCpf1 system by targeting selected genes alone by qRT-PCR, the transcript levels of these genes were significantly reduced compared to the control group (fig. 3B).
Then using WO3The probe further characterizes the extracellular electron transfer capability of the Shewanella cells after the regulation of the CRISPR-ddAsCpf 1. As a result, it was found that the extracellular electron transfer ability of Shewanella bacteria was enhanced as compared with the control bacteria after seven genes among the ten selected gene targets were inhibited by CRISPR-ddAsCpf1 (FIG. 3C). After five of the target genes (sorB, SO0717, fccA, dhc and coxB) were inhibited, the bacteria showed a 1-fold higher extracellular electron transfer rate than the control group, and the enhancing effect of crRNA-coxB was 2-fold higher than the control group. This suggests that the electron current regulation strategy can effectively regulate the electron flux flow direction and make Shewanella bacteria exhibit higher extracellular electron transfer efficiency.
Example 2 CRISPR-ddCpf1 enhanced degradation of contaminants by Shewanella oneidensis MR-1
Materials and methods
1. Bacterial strains, plasmids and culture conditions
The strains, plasmids and culture conditions used in this example were the same as those in example 1.
2. Reduction experiment of contaminants
Hexavalent chromium cr (vi) was selected as a target contaminant for testing the change in contaminant reducing ability of shiva bacteria after the CRISPR-ddAsCpf1 system was enhanced. Contaminant reduction experiments were performed in anaerobic serum vials containing 20ml of sodium lactate mineral salt, with sodium lactate as the only electron donor and the contaminant as the only electron acceptor. The vial was charged with chloramphenicol at a final concentration of 10. mu.g/ml, tetracycline at 5. mu.g/ml and IPTG at 1 mM. Before beginning the contaminant degradation experiment, the resuspended concentrated bacteria were added to the vial, and the bacteria concentration for the Cr (VI) reduction experiment was 1.86 mg bacteria per ml of medium, with an initial concentration for Cr (VI) reduction of 20 mg/L. Cr (VI) reduction experiments were performed in an anaerobic glove box. All experiments were set up in 3 parallel groups.
Second, experimental results
Using cr (vi) as a representative contaminant, we further validated the electron flow regulation strategy to enhance extracellular electron transport capacity and the effect of bioreduction of contaminants. Similar results were obtained in bioreduction experiments for Cr (VI). As shown in fig. 4, we identified 6 potent cr (vi) reduction enhancing genes by CRISPR-ddAsCpf1 system screening, five of which were inhibited with more than 25% increase in cr (vi) reduction in the bacteria (fig. 4A). Whereas, after the dmsE gene was suppressed, the bacterium showed the relatively highest enhancement effect (38.14% increase) in the Cr (VI) reduction rate (FIG. 4B). The genes selected in this example were different from those selected in example 1, which should be due to the difference in electron acceptors. These results indicate that the electron flow regulation strategy was successfully applied to enhance the reduction rate of cr (vi). Thus, this strategy provides an effective way to increase the extracellular electron transport capacity of bacteria, thereby facilitating the reduction of contaminants by the bacteria.
In summary, we have devised a method of using CRISPR-ddAsCpf1 to re-alter the electron flux of Shewanella MR-1 bacteria to enhance their extracellular electron transport capability. In this approach, we first selected potential gene targets based on genetic analysis and previous studies. Second, a crRNA library was constructed to look for gene targets. Third, both crRNA library and the plasmid encoding ddAsCpf1 were transformed into dissimilatory metal-reducing bacteria cells and screened for gene targets with enhanced ability to deliver potent extracellular electrons. In addition to Shewanella MR-1 bacteria, we successfully validated the already developed CRISPR-ddAsCpf1 system for gene silencing in E.coli, thereby indicating that the system can be extended to other bacteria. This electron-flux-steering strategy mediated by the CRISPR-ddAsCpf1 system can also be extended to other dissimilatory metal-reducing bacterial species. Therefore, the method provides an effective platform for exploring the functions of different genes, and lays a foundation for designing dissimilatory metal reducing bacteria by using a synthetic biological tool to carry out more effective environmental remediation applications (such as treating dye-rich wastewater and remedying heavy metal polluted soil and underground water).
The above-mentioned embodiments are intended to illustrate the objects, technical solutions and advantages of the present invention in further detail, and it should be understood that the above-mentioned embodiments are only exemplary embodiments of the present invention and are not intended to limit the present invention, and any modifications, equivalents, improvements and the like made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
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