1. Getting carbon sources from straw
1.1 Aim
China itself is a large agricultural country produces a large amount of straw waste every year, and relevant data show that 865 million tons of straw will be produced nationwide in 2021. The purpose of this experiment is to obtain the carbon source through straw treatment, which can improve the nutrients for the subsequent culture of Corynebacterium glutamicum, realize the overall integrity of the experiment, and also play a role in protecting the environment. The carbon source obtained from straw was mainly glucose obtained through enzyme treatment after treating waste straw with diluted acid.
1.2 Experimental design
Experimental materials
- Straw
- 1% diluted sulfuric acid
- cellulase
- buffer pH4.8 (HAc-NaAc)
Before starting the experiment the straw needs to be pulverized and screened for straw powder 48 mesh. After pretreatment with diluted sulfuric acid, the moisture content in the solids was measured. It can then be treated with cellulase and filtered to obtain glucose.
For more details on the experiments and protocols, as well as the results of the experiments, see the link.
2. Selection of the optimal promoter
2.1 Aim
The purpose of this experiment is to select the promoter which are suitable to play a good role in Corynebacterium glutamicum.
A promoter is a DNA sequence located in the upstream region of a gene, which serves to initiate gene expression during transcription. Optimizing the promoter increases the localization and binding of the CRISPR-MAD7(Cas12a) system on the target gene, thus improving the editing efficiency. By replacing or changing promoters, gene expression levels can be adjusted. By making these improvements, the efficiency of gene editing in Corynebacterium glutamicum by the CRISPR-MAD7(Cas12a) system can be further enhanced.
2.2 Experimental design
Experimental materials
- pEC-XK99E plasmid vector
- Ptuf promoter
- PlacM promoter
- Ptac promoter
- Ptrc promoter
We looked for relevant promoters through the literature. First of all, a general screening was carried out, and we constructed a recombinant plasmid by substituting the promoter on the plasmid of pEC-XK99E. The recombinant plasmid with correct sequencing results was transferred into Corynebacterium glutamicum by electrotransformation, and the suitable promoter were derived by observing the growth of the final plate colony.
For more details about the experiments and protocols, as well as the results of the experiments, please see the links.
3. Selection of the optimal plasmid
3.1 Aim
When using CRISPR-MAD7(Cas12a) for gene editing, it is often necessary to deliver CRISPR components, such as the Cas12a protein and gRNA, into the target cell to ensure that they work properly within the cell. To efficiently deliver CRISPR components into the target cell, it is often necessary to use a plasmid, due to the fact that plasmids can be used to store, replicate and deliver exogenous DNA.
3.2 Experimental design
Experimental material
- pJYS1 plasmid
- pBluescript plasmid
- pXMJ19 plasmid
- gRNA-donor
- MAD7 system
By constructing pJYS1-MAD7-TTTA, pBluescript-MAD7-TTTA, pXMJ19-MAD7-TTTA plasmids, which were transferred into Corynebacterium glutamicum by electrotransformation, the growth of the colonies on the culture medium was observed. The best plasmid can only be determined by checking the situation against each other. Considering the required situation, three BHIS media and the corresponding antibiotics were required for the intra-experimental process.
After repeating the experiment, the most suitable plasmid was selected by observing the growth of the plate colonies as a basis for subsequent experiments.
For more details about the experiments and protocols, as well as the results of the experiments, please refer to the link.
4. Validation of promoters on the optimal plasmid
4.1 Aim
To ensure that promoters capable of efficient expression on the pEC-XK99E plasmid are also highly expressed on the newly determined optimal plasmid, validations of the promoter on the optimal plasmid was performed.
4.2 Experimental design
Experimental Materials
- pJYS1 plasmid
- Ptuf promoter
- PlacM promoter
- Ptac promoter
- Ptrc promoter
The experimental manipulation of the first step was repeated after replacing the plasmid growth of the pEC-XK99E plasmid with the best plasmid.
For more details about the experiments and protocols, as well as the results of the experiments, please see the link.
5. Selection of the best PAM site
5.1 Aim
The purpose of this experiment is to select the PAM site that can help the CRISPR-MAD7(Cas12a) system can work better in Corynebacterium glutamicum.A PAM site is a short sequence adjacent to a target DNA sequence, which is a specific sequence that is required by Cas proteins when they are used to recognize exogenous DNA sequences in the CRISPR-MAD7(Cas12a) system. In the CRISPR-MAD7(Cas12a) system, Cas proteins typically identify a target DNA sequence by recognizing and binding to a PAM site and performing DNA cleavage or other modifications at that location. Different types of Cas proteins have specific PAM preferences, which limits the applicability of the CRISPR-MAD7(Cas12a) system. On top of selecting the pJYS1 plasmid as the basis for applying the CRISPR-MAD7(Cas12a) system in Corynebacterium glutamicum, we will further select the optimal PAM sites in order to improve the efficiency of gene editing.
5.2 Experimental design
Experimental Materials
- pJYS1-MAD7-TTTN plasmid
We firstly selected (Table 1) the PAM sites as experiments through thesis research. By changing the difference of gRNA in the process of plasmid construction, that is to say, changing the difference of PAM sites, and finally linking them together by primer design. The first step is to amplify the target bands by PCR, and then to constitute the recombinant plasmid by one-step cloning method. The recombinant plasmid is transformed into E. coli and verified by colony PCR, then the plasmid is extracted and sent to the company for sequencing. After the plasmid is correct, it can be transformed into Corynebacterium glutamicum by electrotransformation. Finally, the most suitable PAM site can be obtained by comparing the experimental results.
For more details about the experiments and protocols, as well as the results of the experiments, please see the link.
| Selection of the PAM sites |
|---|
| pJYS1-MAD7-gRNA-TTTA(+) |
| pJYS1-MAD7-gRNA-TTTC(-) |
| pJYS1-MAD7-gRNA-TTTT(-) |
| pJYS1-MAD7-gRNA-TTTG(-) |
| pJYS1-MAD7-gRNA-TTTC(+) |
| pJYS1-MAD7-gRNA-TTTA(-) |
| pJYS1-MAD7-gRNA-CTTC(+) |
| pJYS1-MAD7-gRNA-CTTA(+) |
| pJYS1-MAD7-gRNA-CTTG(-) |
| pJYS1-MAD7-gRNA-CTTT(+) |
6. Construct strain of bacteria
6.1 Aim
Through a lot of experiments, we have found the plasmid, PAM site, promoter suitable for use in Corynebacterium glutamicum. In the following experiments, we need to knock out the genes affecting the production of lycopene by using the well established CRISPR-MAD7(Cas12a) system.
We firstly chose to knock out the CrtEb gene, according to the principle that after knocking out CrtEb, it will reduce the further decomposition of lycopene, so as to achieve the purpose of enriching lycopene. And the color of Corynebacterium glutamicum will change from yellow to red after knocking out the CrtEb gene.
6.2 Experimental design
By constructing the plasmid which is the knockdown of CrtEb gene as shown in the figure, the efficiency of the CRISPR-MAD7(Cas12a) system to knockdown the CrtEb gene was verified by transforming the recombinant plasmid which was sequenced correctly into Corynebacterium glutamicum, and observing the growth. And the strain with the correct knockdown gene will be saved as the basis for subsequent experiments. At the same time, we can use the strain that has knocked out the CrtEb gene as a sensory state as a basis for further experiments in the future.
For more details about the experiments and protocols as well as the experimental results, please refer to the link.
7. Build strains that produce high lycopene
Optimizing chassis cells
7.1 Aim
This experiment aims to study the recombinant efficiency by expressing the recE/T enzyme system in C. glutamicum ATCC13032, providing a more efficient chassis for later gene manipulation. Because of the low efficiency of gene recombination for wild Corynebacterium glutamicum. Reviewing the relevant literature, it was found that the expression of recE/T enzyme system significantly improved the recombination efficiency of Corynebacterium glutamicum, and provided a more efficient chassis cell for later gene manipulation.
7.2 Experimental Design
Experimental Materials
- C. glutamicum ATCC13032
- pEC-XC99E-recET
First, plasmids containing genes associated with the recE/T enzyme system were designed and synthesized. Plasmid amplification by transformation of TOP10 strains. The plasmid was then transferred to C. glutamicum ATCC13032 by electroconversion. Positive clones carrying recE/T enzyme system plasmids are obtained by screening for resistance genes (e.g. kanamycin resistance genes). Finally, the sequencing method was used to ensure the successful introduction of recE/T gene.
8. Construction of the ΔcrtEb strain
8.1 Aim
This experiment aims to explore the application of CRISPR-MAD7(Cas12a) system in gene editing by editing the crtEb gene of Corynebacterium glutamicum by using the CRISPR-MAD7(Cas12a) system, and to construct a strain with high yield of lycopene.
8.2 Experimental Design
Experimental Materials
- C. glutamicum ATCC13032-RecE/T
- pJYS1-MAD7-ΔcrtEb
First, plasmids containing genes and gRNAs associated with the CRISPR-MAD7(Cas12a) system were designed and synthesized. The plasmid is then amplified by transformation of TOP10 strains. Next, the plasmid is transferred into Corynebacterium glutamicum cells by electroconversion method, and positive clones carrying the CRISPR-MAD7(Cas12a) system plasmid are obtained by screening for resistance genes (such as kanamycin resistance genes). Gene knockout verification was performed by PCR and sequencing methods to detect the knockout of crtEb gene. Finally, the plasmid is cured by subculture.
9. Determination of lycopene content
9.1 Aim
The aim of this experiment is to measure lycopene yield in Corynebacterium glutamicum using high performance liquid chromatography (HPLC) to evaluate the effect of genetic engineering on lycopene synthesis by setting up a control group.
9.2 Experimental Design
Experimental Materials
M solution. 60%hexane and 40%acetone
-
A solution. 81%methanol,15%MTBE and 4%ddH2O
-
B solution. 7%methanol,90%MTBE and 3%ddH2O
- Take 1 mL of standard product and dry weigh it at 70 degrees Celsius, and obtain 1ml standard with a dry weight of x mg.
- For carotenoid extraction, centrifuge 1 mL of culture. Resuspend the cell pellet in 1 mL M solution with vigorous shaking of the shaker for 60 sec, and transfer the buffer with pigment to a glass tube.
- Repeat the above procedure three times at 4°C until the cell pellet and supernatant are colorless.
- Transfer the supernatant to a brown HPLC vial with a screw cap and filter the sample with a 1 mL syringe and a 0.2 μm sterile organic filter membrane.
- Use [ZORBAX Eclipse, XDB-C18 (particle size 5 μm, inner diameter 4.6 mm, length 250 mm), both of which are used in combination with inverting protection column and diode array detector (DAD) for ultraviolet/visible (Vis) spectral recording]. The α eluate was separated from 100% A solution to 100% B solution by binary gradient elution and the injection volume was 30 μL at a constant flow rate of 1.0 mL/min. Carotenoids were identified by comparing retention time and absorption spectra (410-510 nm) at the 410-510 nm peak λmax, resulting in lycopene yield assays.
10. Engineered cultivation of high-yield lycopene strains
10.1 Aim
The aim of this experiment was to evaluate the growth status and lycopene yield of Corynebacterium glutamicum in fermenter culture, with a view to optimizing fermentation conditions and improving lycopene production efficiency.
10.2 Experimental Design
Experimental Materials
-
Optimized media.37 g/L BHI,91 g/L sorbitol,2 g/L glucose
( Dissolve in deionized water, stir and autoclave)
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Controlled fermenters.The optimal temperature range is 28-32°C. The dissolved oxygen content is controlled at about 30%-50%. The optimal pH range is generally 6.0-8.0.
Corynebacterium glutamicum was cultured in flasks to detect the effects of different media components and concentrations on bacterial growth and lycopene yield. Based on the experimental results, the media formula was optimized. Transfer the optimized medium to a fermenter and inoculate with Corynebacterium glutamicum seed fluid.Set fermentation conditions such as temperature, pH, stirring speed, dissolved oxygen, etc. During fermentation, samples are taken periodically to detect bacterial growth (e.g.biomass, cell density) and lycopene yield. After the end of fermentation, the fermentation broth is collected, and lycopene is extracted and purified by extraction, chromatography and other methods. The yield of lycopene was detected by HPLC and other methods.
