Module 1 Basic fermentation
Cycle1
Design:
In order to enable E. coli engineering strains to produce salidroside, we searched for the biosynthetic pathway of salidroside metabolism and selected one of the two metabolic pathways for modification. L-tyrosine undergoes endogenous transamination to generate 4-hydroxyphenylpyruvate, which is decarboxylated by the pyruvate decarboxylase gene ARO1010 (BBa_K4761000) and then reduced to tyrosol by endogenous ADH (BBa_K4761001). It undergoes glycosylation by the glycosyltransferase UGT85A1 (BBa_K4761002) from Schizonepeta tenuifolia to produce salidroside.
Build:
The pSA plasmid (BBa_K4761100) containing ARO10 gene and ADH gene, the pET plasmid (BBa_K4761101) containing UGT85A1 gene, and the pCS plasmid (BBa_K4761102) containing pgm and galu genes were directly transformed into DH5α using chemical transformation method. In the receptive state, perform colony PCR validation, and initiate fermentation after confirming the absence of errors.
Test:
The results of colony PCR are as shown in Fig1.1 and verified to be correct. Through HPLC experiments, it was found that the standard curve peak had significant fluctuations and cannot be used as a reference.
Learn:
The purity of the standard substance used in this HPLC was insufficient, and after careful literature review, it was found that the standard substance was dissolved in LB culture medium. Afterwards, a higher purity standard of salidroside was used for the preparation of the standard curve. In addition, no control group was set up for this fermentation.
Fig.1 Three plasmid colony PCR results (pSA-ARO10-adh6 validation on left 1-4, pET-UGT85A1 validation on left 5-8, and pCS-pgm-galu validation on left 9-10)
Cycle2
Design:
Prepare the standard curve using a higher purity Rhodiola glycoside standard, dissolve the standard in LB medium, and add a control group (to convert DH5α) Escherichia coli strain.
Build:
According to literature review, the yield of salidroside is approximately in the order of 100 mg/L, and the standard sample is configured with a concentration of 25,50,100,200 mg/L.
Test:
The peak shapes of LB standard with and without salidroside are not significantly different and cannot be analyzed.
Learn:
After reviewing the literature and discussing with the supervisor, it was speculated that the components contained in LB culture medium are very complex, and some of them may be similar to the peak time and shape of salidroside. Therefore, water and M9 culture medium will be used as solvents for making standard curve next time.
Cycle 3
Design:
Use water and M9 medium to prepare the standard solution, with a concentration gradient set to 25,50,100,200mg/L.
Build:
Consistent with cycle 2 operation
Test:
Successfully obtained the standard curve, as detailed in Fig.2 and Fig.3. Fig.2 used water as the solvent, and Fig.3 used M9 medium as the solvent.The fermentation results of pSA-ARO10-adh6, pET-UGT85A1 plasmid strain are shown in Fig.4, and the fermentation results of pSA-ARO10-adh6, pET-UGT85A1, pCS-pgm-galu plasmid strain are shown in Fig.5. See the Results module for detailed analysis.
Fig.2 Standard curve made with water as solvent (a: 0mg/L; b: 25mg/L; c: 50mg/L; d: 100mg/L; e: 200mg/L)
Fig.3 Standard curve made with M9 medium as solvent (a: 0mg/L; b: 25mg/L; c: 50mg/L; d: 100mg/L; e: 200mg/L)
Fig.4 Fermentation results of pSA-ARO10-adh6, pET-UGT85A1 plasmid strains (a: 24h; b: 36h)
Fig.5 Fermentation results of pSA-ARO10-adh6, pET-UGT85A1,pCS-pgm-galu plasmid strains (a:12h;b: 24h; c: 36h)
Module 2 Copy number optimization
2.1 Triple Plasmid Skeleton Gene Matching
Cycle1
Design:
To achieve an increase in the unit yield of salidroside and optimize the configuration of metabolic pathways, we designed a strategy to regulate metabolic pathways by altering the copy number of exogenous genes. Three types of plasmids were used: pSA (BBa_K4761100) with low copy number, pET (BBa_K4761101) with high copy number, and pCS (BBa_K4761102) with medium copy number. We constructed six new plasmids by pairing specific genes with these plasmid skeletons. The gene-skeleton junctions commonly utilized KpnI and BamHI cleavage sites. Our plan involved enzymatic cleavage of the plasmid skeletons, gel recovery, and subsequent ligation to construct the new plasmids. However, due to the small size of genes and skeleton fragments on pSA and pCS plasmids, electrophoresis alone was not effective in separating them. To overcome this, an additional enzyme digestion step was introduced. If the skeleton was recovered, the genes were subjected to enzyme digestion, and vice versa. Please refer to Figure 6 for a detailed digestion strategy.
Build:
use the above design to carry out the corresponding digestion. Once the digestion is completed, proceed with gel electrophoresis to separate the fragments, followed by enzyme ligation.
Test:
It was found that the pSA plasmid cleaved by multiple enzymes showed no bands in the pCS plasmid electrophoresis results, while the pET plasmid showed clear and correct size bands. The electrophoresis results after enzyme digestion are detailed in Figure 7.
Learn:
Analyze the reasons and consider whether the simultaneous use of multiple endonucleases affects the efficiency of excision, as the system using three enzymes has no bands while the system using two enzymes has bands. Therefore, two separate enzyme digestion methods were used for pSA and pCS plasmids.
Fig.6 Restrictive endonuclease selection strategy
Fig.7 Electrophoresis results of three restriction endonucleases (from left to right, consistent with Fig.6 from top to bottom)
Cycle2
Design:
Two different combinations of endonucleases were used to cleave pSA and pCS plasmids, to investigate whether the simultaneous use of multiple endonucleases affects the efficiency of each other.
Build:
Configure five systems as follows: Left 1, 2, and 3 with pSA plasmids and KpnI, BamHI, respectively; KpnI and BsaI; BamHI and HindIII. Systems 3 and 4 with KpnI and BamHI of pCS plasmids; KpnI and SpeI.
Test:
There are still no obvious bands, as shown in Figure 8.
Learn:
It indicates that multiple enzymes do not affect each other's efficiency. After discussion, it was suggested that the less conspicuous electrophoresis results may be attributed to the low copy numbers of pSA and pCS plasmids. Because pSA is a single copy plasmid. Use the method of concentrating plasmids to increase the concentration of plasmids, and then perform enzyme digestion operations.
Fig.8 Electrophoresis results after two enzyme digestion methods
Cycle3
Design:
Amplify the bacterial solution from multiple test tubes, ultimately extract plasmids from 8-10 tubes, and then use a gene cleaning kit for concentration operation.
Build:
Concentrate according to the operation manual of the gene cleaning kit, perform enzyme digestion, and run the gel.
Test:
Still no obvious bands appear.
Learn:
Abandon the simple enzyme digestion and enzyme ligation operation, and use PCR, enzyme digestion, and enzyme ligation methods to reconstruct the plasmid.
Cycle4
Design:
Design primers located at both ends of the gene and skeleton, paying attention to including cleavage sites and adding protective bases at both ends of the cleavage sites. The PCR primer sequence can be found in primer numbers 1-10.
Build:
Using the original three plasmids as templates, six fragments were PCR generated, including three genes and three skeletons. Gel electrophoresis was performed to verify whether the stripe size was correct (see Fig.9 for details). After transformation, colony PCR validation was performed to verify that one segment of the primer was on the gene and the other segment was on the skeleton. The specific sequence is shown in primer numbers 1-11 to 1-16, and the correct band should be 1200 (see Fig.10 and Fig.11 for details).
Test:
The reconstructed plasmids pSA-UGT85A1 and pET-ARO10-adh6 were successful, while the reconstructed plasmids pSA-pgm-galu and pCS-UGT85A1 showed significant heterobands. Further sequencing verification failed.
Fig.9 Electrophoresis results of three plasmids after PCR (from left to right, 1 is the pSA skeleton, 2 is the ARO10-adh6, 3 is the pET skeleton, 4 is the UGT85A1, 5 is the pCS skeleton,
Fig.10 Reconstructed Plasmid Colony PCR Results (pSA-UGT85A1 on left 1-4, pSA-pgm-galu on left 5-7)
Fig.11 Reconstructed Plasmid Colony PCR Results (Left 1 is pSA-pgm-galu, Left 2-5 is pET-ARO10-adh6, Left 6-9 is pET-pgm-galu, Left 10-13 is pCS-ARO10-adh6, Left 14-17 is pCS-UGT85A1)
2.2 High copy plasmid skeleton replacement
Cycle1
Design:
Obtain three plasmids containing dual T7 promoter expression boxes, pACYCDuet (BBa_K4761103), pETDuet (BBa_K4761104), and pCDFDuet (BBa_K4761105). Use gibson assembly to insert the ARO10 and adh6 genes into the first T7 expression box, and the UGT85A1 gene into the second T7 expression box. The expression level of the second T7 expression box is relatively high. To express these three plasmids, they need to be transferred to the BL21 (DE3) strain. Assemble two genes into the plasmid skeleton in a step-by-step manner.
Build:
Use gibson assembly to integrate the ARO10-adh6 gene into the first T7 expression box, design primers, and refer to primer numbers 1-17 to 1-26 for primer sequences. The Tm value of the homologous sequence is 55 ℃. First, use PCR to obtain a linearized vector fragment with homologous fragments and the gene fragment. After digestion by DpnI, perform electrophoresis gel run to verify the correct size of the band, and then proceed with the assembly reaction.
Test:
The results of the electrophoretic gel run after DpnI digestion are correct (refer to Fig. 12 for details), but the transformed validation bacteria, p results, are incorrect. The validation primers are indicated by primer numbers 1-32 to 1-39, and there are multiple heterobands visible, as shown in Fig. 13. The target 1000bp band is present but not very pronounced.
Learn:
It is speculated that the bacterial strain is impure, continue to line culture, and expect to obtain a single colony.
Fig.12 Electrophoresis results after linearization of vector genes
Fig.13 Colony PCR Results
Cycle2
Design:
Purification of bacterial colonies with many mixed bands is expected to obtain a single band.
Build:
After liquid expansion culture of the colony, a single colony is drawn.
Test:
It was found that there were still heterozygous bands in the gel. After sequencing, the results indicated that the genes were not integrated into the genome.
Learn:
Redesign primers, set the Tm value of homologous fragments to 60 degrees, and observe the results again through experiments.
Cycle3
Design:
Increasing homologous fragments may enhance assembly efficiency. Additionally, the ugt85a1 gene was selected for integration, as the smaller fragment may result in a higher success rate of integration.
Build:
Increase the Tm value of the homologous fragment from 55 ℃ to 60 ℃, with the remaining conditions unchanged. The new primer sequence can be found in primer numbers 1-27 to 1-31.
Test:
Two plasmids, pacyc-UGT and pETDuet-UGT, were successfully constructed through sequencing identification. The sequencing comparison results are shown in Fig.14 and Fig.15, and the construction of the pCDFDuet plasmid failed.
Learn:
The Tm value of homologous fragments has a significant impact on integration efficiency.
Fig.14 Comparison of PacycDuet UGT85A1 Sequencing Results
Fig.15 Comparison of pETDuet-UGT85A1 sequencing results
Cycle4
Design:
After successfully integrating the UGT gene into the second expression box of the plasmid, the ARO10-adh6 gene was integrated into the first expression box, and two pairs of primers were used for validation in colony PCR validation to prevent the loss of the UGT gene. The primer sequences are shown in primer numbers 1-32 to 1-39.
Build:
Similarly, the ARO10-adh6 gene was integrated using gibsonassembly, using a new primer designed for loop 3 to obtain a linearized vector and gene fragments with homologous sequences.
Test:
Using two pairs of primers, it was found that the PCR electrophoresis bands of the colonies were all correct, as shown in Fig.16, and the sequencing results were also correct.
Fig.16 Colony PCR electrophoresis verification after two Gibson reactions (left 1-8 for UGT85A1 gene verification, right 1-8 for ARO10-adh6 gene verification)
Module 3 Gene knockout (pheA, pykA, pykF three gene knockout)
Cycle1
Design:
We plan to use CRISPR-Cas9 for gene knockout and prepare Donor fragments containing both ends of the target gene, achieving seamless knockout through homologous recombination. Two plasmids are involved, namely pTarget (BBa_K4761107) and pCas plasmid containing Cas9 gene (BBa_K4761006), which can express sgRNA.
Build:
Design sgRNA using an online website (CHOPCHOP https://chopchop.cbu.uib.no) and obtain pTarget plasmids containing the target sgRNA through rPCR. The primer sequences are shown in primer numbers 2-13, 2-14, 2-19, 2-20, 2-25, 2-26. Perform further transformation and sequencing after transformation.
Test:
The sequencing results showed that it was the original plasmid sequence, but the target sgRNA sequence could not be replaced through rPCR.
Learn:
When using circular plasmids as templates, use DpnI enzyme digestion, otherwise false positives may occur.
Cycle2
Design:
Add DpnI digestion step after rPCR preparation of new pTarget to remove the original template.
Build:
Take 26ul of PCR product, add 3ul Buffer and 1ul DpnI enzyme, and react for 1.5h at 37 ℃.
Test:
The sequencing results are correct.
Cycle3
Design:
Prepare the receptive state of bacteria containing pCas plasmid and transform the pTarget plasmid containing target sgRNA into the receptive state containing pCas together with Donor.
Build:
Using the original strain as a template, Donor was prepared through one PCR and one fusion PCR. The primer sequences are shown in primer numbers 2-1 to 2-12. The pTarget plasmid and Donor were transferred using chemical transformation method and cultured in a 30 ℃ incubator.
Test:
After transformation, a large number of colonies grow on the plate, making it difficult to easily select single colonies. In addition, there was no band during the validation of the primers, as shown in Fig.17.
Learn:
A large number of colonies have grown, indicating that the concentration of pCas containing plasmid responsive cells was too high during preparation, and the amount of transformation used was appropriately reduced afterwards. During the validation of electrophoresis, there was a lack of bands, and upon examination, it was found that the validation primers should be designed on the genome.
Fig.17 Gene Knockout Verification Results (left side indicates not knocked out, right side has no bands)
Cycle4
Design:
Reduce the amount of pCas receptive states during conversion and redesign the validation primers.
Build:
Search for the location of the target knockout gene on the NCBI website and design validation primers at approximately 1000 bp upstream and downstream, as shown in primer numbers 2-33 to 2-38.
Test:
It was found that there were still 3000 bands during electrophoresis verification, indicating that the gene was not knocked out, as shown in Fig.18.
Learn:
The efficiency of sgRNA varies, and different sgRNAs are redesigned using online websites for knockout experiments.
Fig.18 Gene Knockout Verification Results (New Primer)
Cycle5
Design:
Knockout efficiency is related to the sequence of sgRNA, so multiple different sgRNA sequences are selected to study knockout efficiency.
Build:
After designing the online website, the pTarget plasmid was re prepared, with primer sequences listed in primer numbers 2-13 to 2-32 for transformation.
Test:
The target gene was successfully knocked out, and after comparison with the control group, the band was verified to have changed from 3000bp to 2000bp, indicating a perfect replacement. See Fig.19, Fig.20.
Learn:
We initially selected the sgRNA with the highest theoretical knockout efficiency on the website, but the final result was that the sgRNAs ranked lower in theoretical efficiency successfully knocked out the target gene. Therefore, when selecting relevant sgRNAs, the number of groups should be increased.
Fig.19 pheA gene knockout verification (successful knockout of left 1, left 3, and left 5)
Fig.20 pykF gene knockout verification (successful knockout of left 1, left 3, left 6, and left 7)
Module 4 fusion expression
Cycle1
Design:
Three flexible and three rigid connectors (BBa_K4761030-BBa_K4761035) were designed to connect adh6 and ARO10 genes. The fusion expression essence is that the stop codon of the first gene is replaced by the linker sequence. Since these two genes were originally on the pSA (BBa_K4761100) plasmid skeleton, pSA was chosen as the plasmid skeleton.
Build:
The two genes were amplified by primer PCR with primer numbers 3-1 to 3-14, and then the two genes linked by linker were obtained by fusion PCR. Then the recombinant plasmid was obtained by enzyme-cutting and enzyme-linking, and then chemically transformed into the receptive state of Escherichia coli.
Test:
Use two primers 3-15 and 3-16; For the single colonies obtained by bacteria 3-15 and 1-14 p verification, the bands obtained should be 1000 and 3000 bands, but the results were mixed (see Fig.21), and the bands were not correct at the same time, so sequencing was carried out, and the sequencing results were wrong, showing self-cyclization.
Learn:
Because the host vector used is pSA plasmid, which is single copy, the extracted plasmid concentration is low, and the efficiency of enzyme digestion and enzyme-linking is low, other high-copy plasmid scaffoldings are considered as vectors for fusion protein expression.
Fig.21 Colony PCR electrophoresis verification results (using pSA as the skeleton)
Cycle2
Design:
Select the high copy plasmid skeleton pET (BBa_K4761101) as the vector for fusion protein expression and observe the effect.
Build:
After fusion expression, sequence the PCR product to determine if the linker sequence is correct. Other experimental procedures are the same as those in cycle 1.
Test:
The sequencing results of the PCR product after fusion expression are partially correct. Colony PCR validation was performed on the transformed colonies, and the bands were correct. The sequencing results are also correct, as shown in Fig.22.
Learn:
When constructing a new plasmid using enzyme-linked cleavage, the concentration of the plasmid has a significant impact on the experimental results, and it is necessary to ensure a high concentration of the plasmid skeleton.
Fig.22 Comparison of pET-adh6-ARO10 Reconstructed Plasmid Sequencing
Module 5 Gene integration
Cycle1
Design:
We plan to integrate the ARO10 gene and UGT85A1 gene into Escherichia coli using CRISPR RNA guided integrations. This system involves three plasmids: pEffector (BBa_K4761052), pDonor (BBa_K4761108), and pCutamp. The pEffector plasmid contains specific sgRNA sequences that determine the location of gene integration. The pDonor plasmid can amplify foreign gene fragments in organisms, and pCutamp contains genes such as Cas9.
Build:
We integrated the ARO10 gene and UGT85A1 gene into the pDonor plasmid using the Gibson assembly method between the Transpson Left End (BBa_K4761050) and Transpson Right End (BBa_K4761051). According to the plasmid map, we designed primers to linearize the vector, with primer numbers 4-1 to 4-6. Leave a distance between the primer and two transposable elements (Transpson Left End, Transpson Right End).
Test:
After linearizing the vector, electrophoretic verification was performed and it was found that the band was around 5000, which is the first lane on the left of Fig.23. The correct value should be around 3000, so sequencing was performed.
Learn:
The sequencing results showed that there was an unknown sequence between the two transposable elements, resulting in a much larger linearized vector fragment than expected. The primers were redesigned onto the two transposable elements to ensure that no excess fragments were inserted.
Fig.23 Electrophoresis results of linearized vector genes (left 1 is the pDonor plasmid skeleton, left 2 is ARO10, and left 3 is UGT85A1)
Cycle2
Design:
Using newly designed primers numbered 4-7 to 4-12, linearize the pDonor vector and two genes.
Build:
The experimental method is the same as cycle 1.
Test:
The results of pDonor linearization carrier electrophoresis are correct, as shown in Fig.24, which is around 3000. The validation results of transformed colony PCR are correct, and the colony PCR primer numbers are 4-13, 4-14, 4-23.
Learn:
Proficient in analyzing the reasons in the intermediate process, and should first test and verify the correctness when using unknown plasmids given by others.
Fig.24 Electrophoresis results of the linearized vector gene with a new primer (right 1 is the pDonor plasmid skeleton, right 2 is ARO10, and right 3 is UGT85A1)
Cycle3
Design:
After the construction of the pDonor ARO10 and pDonor UGT85A1 plasmids, they were transferred into Escherichia coli DH5α along with the plasmids pEffector and pCutamp, And after sufficient cultivation time, we hope that the corresponding gene can be inserted into the correct position in the genome.
Build:
Three plasmids were transferred into Escherichia coli using chemical transformation methods.
Test:
After conducting colony PCR validation, we successfully identified E. coli DH5α The three plasmids mentioned above were transferred and the corresponding genes were successfully inserted into the correct positions in the genome, as shown in Fig.25.
Fig.25 Gene Integration Electrophoresis Results
Module 6 Liposome vesicle
Cycle 1
Design:
We first plan to prepare simple liposome vesicles to verify the accessibility of liposomes and to explore the appropriate experimental conditions for the preparation of liposome vesicles.
Build:
Liposome vesicles were prepared in a rotary evaporator using the thin-film rehydration method with water for hydration.
Test:
At the end of the preparation, the results were characterized by dynamic light scattering and optical microscopy, and it was found that monolayers and multilayers of liposome vesicles were successfully prepared (showed in Fig.26).
Learn:
The thin film rehydration method can be a viable method for liposome vesicle preparation, although the specific conditions may need to be further explored.
Fig.26 Optical microscope result of lipsome hydrated with water, PC:CH=7:2
Cycle 2
Design:
After the successful preparation of liposome vesicles, we plan to prepare liposomes capable of encapsulating E. coli.
Build:
The same preparation method was used as Cycle 1, but the water in hydration was replaced with LB liquid medium containing E. coli.
Test:
The characterization method was the same as Cycle 1, and it turned out that in addition to monolayer and multilayer liposome vesicles, some of the liposome vesicles could be found wrapping E. coli in them under the optical microscope to achieve bacteria loading (showed in Fig.27).
Learn:
After replacing water with LB medium, the thin-film rehydration method can still successfully prepare liposome vesicles, although some experimental conditions need to be fine-tuned (such as rotational speed of the rotary evaporator, etc.)
Fig.27 Optical microscope result of lipsome hydrated with LB medium containing E.coli, PC:CH=7:2
Cycle 3
Design:
We would like to investigate the effect of different ratios of cholesterol and phosphatidylcholine on the formation of liposomes.
Build:
We set up several sets of different ratios of cholesterol and phosphatidylcholine as raw materials, using the same preparation method as in Cycle 2.
Test:
The characterization method is the same as Cycle 1, and we found that the liposomes were best prepared when the mass ratio of cholesterol and phosphatidylcholine was 7 to 2 (showed in Fig.27, Fig.28 and Fig.29).
Learn:
Experiments were conducted using controlled variables to obtain the best ratio of ingredients.
Fig.28 Optical microscope result of lipsome hydrated with LB medium containing E.coli, PC:CH=7:4
Fig.29 Optical microscope result of lipsome hydrated with LB medium containing E.coli, PC:CH=7:6