Engineering Success
Construction of the pMTL-ΔperR plasmid
[DESIGN]
PerR or its homologous protein is the transcriptional repressor involved in the H2O2 stress response in Firmicutes, which deletion is reported to enable a rapid induction of genes required to protect cells from a H2O2 stresses.
The role of the PerR repressor has been studied in C. acetobutylicum where it is proposed that this regulator acts as a switch to O2 tolerance, and the genes encoding the components of the alternative detoxification system were found to be PerR regulated. While a wild-type C. acetobutylicum strain loses viability after exposure to H2O2 or molecular oxygen, the inactivation of the PerRCA repressor leads to a better resistance to H2O2 as observed in other Firmicutes.
We want to analyze the growth of C. tyrobutyricum after knocking out perR in its genome, so we constructed this plasmid for the deletion of perR.
Fig-1 The expression vector of the pMTL-ΔperR plasmid
[BUILD]
The upper and lower homology arms (HR-UP, HR-DOWN) were constructed at about 500 bp upstream and downstream of the PerR sequence of the C. tyrobutyricum genome, plus a lactose-inducing promoter and a spacer sequence derived from the PerR , generating the CRISPR array . Using the plasmid pMTL82151 from C. tyrobutyricum as a template, a linear vector was obtained by EcoRI and KPnI double digestion. The CRISPR array and linearized vector after double digestion were ligated using Gibson's assembly method. Colony PCR is performed on the transformed colonies. The positive colonies with correct colony PCR were transferred and the plasmid was picked, and the recombinant plasmid was obtained after sequencing verification: pMTL-perR-HR.
[TEST]
The growth profiles of the plasmid knockout strain under different contents of oxygen
After the experimental strain was activated by TGA plate medium, the single colony was selected and incubated at 37 °C in TGA liquid medium until the OD600 was about 1.0 as fermentation seed solution, and transferred to a 50 mL volume centrifuge tube containing 15 mL TGA medium according to the 5% volume specific inoculation amount, and placed in 37 °C 0r, 50 r, 100 r fermentation for 80 h respectively for product detection.
Colony morphology observation
After the experimental strain was activated by TGA plate medium, the single colony was selected and incubated at 37 °C in TGA liquid medium until the OD600 was about 1.0 as seed solution, and transferred to a 50 mL volume centrifuge tube containing 15 mL TGA medium according to the 5% volume specific inoculation amount, and placed in 37 °C 0r, 50 r, 100 r cultivate for 80 h respectively for growth detection.
Results
As can be seen from the sequencing results, the perR fragment has been knocked out.
Fig 2. Sequencing verification of obtained mutant. The red part represents the absence of the sequence.
At different rotational speeds, the growth of the plasmid knockout strain was better than that of the control strain.
Fig 3. The growth profiles of Ct (ΔperR) and Ct (Control) under different contents of oxygen
Fig 4. Physiological characteristics of Ct (ΔperR) and Ct (Control) in the presence of oxygen. (A) The intracellular ROS levels in Ct (ΔperR) and Ct (Control) in the presence of oxygen. ROS levels were measured in the logarithmic prophase. (B) The ratio of NADH/ NAD+ in Ct (ΔperR) and Ct (Control) in the presence of oxygen. Phase I, II and III refer to the early-, mid- and late- logarithmic phase, respectively.
Fig 5. The survival rate of Ct (ΔperR) and Ct (Control) to oxidative stress.
[Learn]
In the experiments, we found that the knockout plasmid was still present in C. tyrobutyricum after completing the knockout of perR , which somewhat hindered the introduction of the subsequent plasmids. In reviewing the literature we learned that, tetR is a repressant. In the absence of tetracycline, tetR binds to the tetracycline operon to represses the transcription of the tetracycline overflow pump gene. When tetracycline enters the cell, tetracycline binds to tetR and changes its conformation, then tetracycline operon is not hindered and can express the CRISPR array behind it normally，thus knocking itself out. Therefore, we hope to eliminate the imported plasmids by adding the tetR fragment to the plasmid and then by tetracycline induction at a later stage.
Construction of the Plac->ΔperR -> self - target plasmid
[DESIGN]
As the previously constructed pMTL-ΔperR plasmid has been introduced into C. tyrobutyricum, introducing other plasmids like pMTL-Pvgb-perR could be difficult, and the growth of the bacteria would be impacted, so we expected that the plasmid using for knocking out perR could be degraded after completing its task, and that leads us to the design of the Plac->ΔperR->self-target plasmid.
The tetR gene encodes the tetracycline repressor protein(TetR). And in the absence of tetracycline, it binds to the tetracycline operon(tet operon, tetO) to inhibit the transcription of the plasmid self targeting CRISPR array. When tetracycline is added, TetR binds to tetracycline instead, and the transcription of downstream CRISPR array would be carried out and the plasmid will be degraded.
Fig-6 The expression vector of the Plac->ΔperR -> self - target plasmid and its mechanism
[BUILD]
Use the previously constructed pMTL-ΔperR as a template to obtain pMTL-ΔperR linearized vector by PCR amplification, and extract C. tyrobutyricum genome as a template to amplify the tetR fragment with homologous arms. Then the tetR fragment and the pMTL-ΔperR linearized vector were connected using Gibson-assembled method by homologous recombination. Colony PCR was performed on the transformed colonies, and the positive colonies with correct agarose gel band were transferred and cultured. The plasmids were extracted for sequencing verification, and correct plasmids would be obtained.
[TEST]
Characterization experiment
Our plasmid constructed contained methsulfamycin resistance gene for inducing knockdown of the perR gene with 40mmol/L lactose in RCM medium at 3μl/mL Tm and 2.5ul/mL D-cycloserine, after induction of 8-10 generations, the coating plate is diluted and the single colony is subjected to colony PCR to verify that perR is successfully knocked out. After successful knockout of the perR on the genome, followed by a certain concentration of tetracycline addition in non-resistant RCM medium. After induction 8 – 10 generations, we obtained single colonies by dilution coated plate for colony PCR validation.
Fig 2. Plasmid-self-target bacteria colonies PCR results
The results show that the target plasmid colonies PCR results appear band to eliminate the plasmid bacteria no band, at the same time, eliminate the target plasmid bacteria cannot survive in the medium containing resistance, prove that the target bacteria has completed plasmid elimination.
We hope to induce the elimination of plasmids by tetracycline after lactose-induced knockout of the gene of interest, and at present, we have only achieved lactose-induced knockout of the gene of interest with success. In addition, we designed plasmids solely to eliminate plasmids to verify whether the method of eliminating plasmids using tetr is feasible in C. tyrobutyricum. We introduced a plasmid carrying the tetR gene in C. tyrobutyricum and successfully eliminated it by tetracycline induction, demonstrating that this method is feasible. The work of knocking out the gene of interest and then eliminating the plasmid for time reasons is not completed.
[Learn]
In the early experiments using lactose inducible promoter to regulate the expression of genes, but in the actual process, especially perR gene knockout process, we found that although has induced many generations, but always cannot induce gene expression, in the literature we learned that because we choose RCM medium contains a high proportion of glucose, and glucose and lactose will produce fusion phenomenon so that lactose can not play a good role on our gene induction. This is a large part of the slowness of our experiments. We then wished to perform induced knockdown by the xylose promoter that does not undergo a fusion phenomenon with glucose.
Construction of the pMTL-Pthl-perR plasmid
[DESIGN]
The growth data of C. tyrobutyricum when perR gene is not expressed can be obtained by constructing plasmid with knock-out perR gene and introducing it into C. tyrobutyricum. We also hope to find out whether excessive expression of perR gene will inhibit the growth of flora compared with wild C. tyrobutyricum, so we choose to use constitutive promoter Pthl to over-express perR gene and observe the growth of flora after expression.
[BUILD]
The main chain pMTL82151 of plasmid was digested by BamHI enzyme, and the linear vector was obtained. Using C. tyrobutyricum genome as template, PerR gene fragment was amplified by PCR.
Gibson assembly method was used to link PerR gene fragment with linearized pMTL82151 vector. The transformed colonies were detected by colony PCR. The correct (positive) colony was cultured for plasmid extraction, and verified by nucleotide sequencing to obtain the recombinant plasmid: pMTL-Pthl-perR.
[TEST]
The growth profiles of the Engineered strains under different contents of oxygen
After the experimental strain was activated by TGA plate medium, the single colony was selected and incubated at 37 °C in TGA liquid medium until the OD600 was about 1.0 as seed solution, and transferred to a 50 mL volume centrifuge tube containing 15 mL TGA medium according to the 5% volume specific inoculation amount, and placed in 37 °C 0r, 50 r, 100 r cultivate for 80 h respectively for growth detection.
Fig 4- Physiological characteristics of Ct (Pthl-perR) and Ct (Control) in the presence of oxygen.
[LEARN]
We found that by knockout of perR gene, C. tyrobutyricum growth is inhibited under anaerobic conditions, and the overexpression of perR gene C. tyrobutyricum is inhibited in growth under aerobic conditions, so we wanted to regulate perR gene expression by the oxygen-inducible promoter, and then C. tyrobutyricum has higher oxygen resistance.
Construction of the pMTL-Pvgb-Bs2 plasmid
[DESIGN]
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 C. tyrobutyricum by detecting the fluorescent expression intensity of fluorescent protein Bs2.
[BUILD]
The linear vector was obtained by single digestion of plasmid backbone pMTL82151 using Bam HI enzyme. The recombinant plasmid pMTL-Pthl-Bs2 offered by Dr. Zhengming Zhu’s research group was used as the template to generate the Bs2 fragment. The Bs2 fragment was then used as template and promoter vgb sequence was constructed upstream by PCR technique to obtain fragment Pvgb-Bs2, meanwhile a pair of homologous arms was added both side for recombination.
The Pvgb-Bs2 fragment and the pMTL82151 linearized vector were linked by Gibson assembly method, and colony PCR was performed on the transformed colonies. The correct(positive) colonies were cultured for plasmid extraction, and those plasmid were verified by nucleotide sequencing to obtain the recombinant plasmid: pMTL-Pvgb-Bs2.
[TEST]
Fluorescence analysis
Due to the tight time we chose to verify the promoter in E. coli first.The bacteria introduced with 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. Two milliliters of bacterial culture solution were taken, centrifuged at 12,000 rpm for 2min, washed with 700ul 1xPBS buffer for 2 times. After resuspending the bacterial pellet using another 700ul 1x PBS buffer, 200 μl of that solution were pipetted into a 96-well plate for microplate reader analysis, in which the obtained data was analyzed.
Result
The test group: the fluorescence intensity is relatively high
The control group: the fluorescence intensity is lower compared to the test group
[learn]
We wanted to express Bs2 fluorescent protein by using the oxygen- inducible promoter vgb, and used the expression of fluorescence intensity to verify its situation under oxygen regulation in C. tyrobutyricum. Due to limited time, we did its expression in E. coli, and the data showed that its fluorescence intensity in microoxygen is much greater than that in high dissolved oxygen amount. Therefore, in subsequent experiments, we replaced the strong promoter thl with this promoter
Construction of the pMTL-Pvgb-perR plasmid
[DESIGN]
Pvgb is an oxygen-dissolving regulated promoter from Vitreoscilla. We have done its expression effect in C. tyrobutyricum before, which proves that it can express bs2 fluorescent protein in different amounts under different oxygen concentrations of C. tyrobutyricum. On the premise that the expression of perR gene can affect the growth of C. tyrobutyricum, We hope that pvgb promoter can be connected with perR gene by Gibson assembly method, and perR gene can be expressed in different degrees by pvgb promoter and the conditions of controlling different oxygen concentrations, so that C. tyrobutyricum has higher oxygen adaptability.
[BUILD]
Using the recombinant plasmid pMTL-Pthl-Bs2 provided by Dr. Zhu Zhengming's research team as a template, the pMTL-Pvgb linearized vector was amplified. With pMTL-Pthl-perR as template, the PerR gene fragment was amplified. Gibson assembly method was used to link PerR gene fragment with pMTL-Pvgb linearization vector. And the transformed colonies were detected by colony PCR. The correct (positive) colony was cultured for plasmid extraction, and verified by nucleotide sequencing to obtain the recombinant plasmid: pMTL-pvgb-perR.
[TEST]
The growth profiles of the Engineered strains under different contents of oxygen
After the experimental strain was activated by TGA plate medium, the single colony was selected and incubated at 37 °C in TGA liquid medium until the OD600 was about 1.0 as seed solution, and transferred to a 50 mL volume centrifuge tube containing 15 mL TGA medium according to the 5% volume specific inoculation amount, and placed in 37 °C 0r, 50 r, 100 r cultivate for 80 h respectively for growth detection.
[Expect]
In subsequent experiments, we will further test the effect of dynamic regulation of perR by Pvgb on the growth of C. casitirate, and we expect to achieve better growth than wild bacteria in both microoxygen and aerobic conditions.