1. Experiment Phase 1: Construction of engineered strains containing GPS, CsTPS1, and GeDH.
Plasmid Recombination and Transformation Experiment 1
Objective: Using pET28a, pET21a, and pBAD33 as vectors, the amino acid sequences of the three enzymes, GPS, CsTPS1, and GeDH, were designed into three recombinant plasmids respectively to facilitate subsequent strain screening and target protein expression.
Design of High-Copy Recombinant Protein Plasmids 01/07/2025 Tuesday
Objective: Optimize the sequences of the three enzymes (GPS, CsTPS1, and GeDH) and insert these gene sequences into the vector plasmid pUC19 using restriction enzyme digestion and ligation methods to obtain high-copy recombinant protein plasmids. These sequences were designed in SnapGene.
Results:
Fig.1 Recombinant plasmid designs for pUC19-GPS, pUC19-CsTPS1 and pUC19-GeDH
Participants: Ethan Wu, Boyan Zhang
Design of High-Expression Recombinant Protein Plasmids 01/07/2025 Tuesday
Objective: Using pET28a, pET21a, and pBAD33, three high-expression plasmids, as vectors, the amino acid sequences of the enzymes GPS, CsTPS1, and GeDH were utilized to construct recombinant protein expression systems via seamless cloning techniques, aiming to achieve higher target protein expression levels.These sequences were designed in SnapGene.
Results:
Fig.1 Recombinant plasmid designs for pUC19-GPS, pUC19-CsTPS1 and pUC19-GeDH
Participants: Ethan Wu, Boyan Zhang
Obtaining the High-Copy Recombinant Protein Plasmids 08/07/2025 Tuesday
Objective: The high-copy protein plasmid we designed is stored in E. coli. To obtain the target gene fragments, it needs to be extracted.
Results: The name and concentration of the plasmids are shown in Table 1.
| Name | Concentration (ng/μl) |
|---|---|
| pUC19-GPS | 342.05 |
| pUC19-CsTPS1 | 281.00 |
| pUC19-GeDH | 294.83 |
Participants: Ruiyang Wen, Yanzhao Liu
Obtaining the target gene fragments 08/07/2025 Tuesday
Objective: The fragments of GPS, CsTPS1, and GeDH genes were obtained through restriction enzyme digestion from the pUC19 plasmid designed in the previous experiment. Subsequently, PCR amplification was performed for the subsequent Gibson cloning.
Results: Through the experiment, we expect to have obtained fragments of the three target genes. The results will be further confirmed through agarose gel electrophoresis and sequencing. The concentrations of the three target gene fragments after PCR amplification are shown in Table 2.
| Name | Concentration (ng/μl) |
|---|---|
| GPS | 205.18 |
| CsTPS1 | 199.72 |
| GeDH | 186.13 |
Participants: Ruiyang Wen, Yanzhao Liu
Obtaining the vectors 08/07/2025 Tuesday
Objective: In order to assemble the recombinant protein plasmids, we need to design primers targeting appropriate sites on the three plasmids, pET28a, pET21a, and pBAD23, and perform PCR amplification to obtain sufficient vector for Gibson cloning.
Results: The concentrations of the three plasmids after PCR amplification are shown in Table 3.
| Name | Concentration (ng/μl) |
|---|---|
| pET28a | 172.66 |
| pET21a | 169.45 |
| pBAD23 | 177.53 |
Participants: Ruiyang Wen, Yanzhao Liu
Validation of vector and target gene 07/08/2025 Tuesday
Objective: Preliminary confirmation of vector and target gene acquisition using DNA agarose gel electrophoresis. Preparing for the next step of Gibson cloning.
Results: Preliminary validation of the obtained vector and target gene fragments was performed using DNA agarose gel electrophoresis. The sizes of GeDH, CsTPS1, and GPS fragments are 1266 bp, 1665 bp, and 1158 bp, respectively. The sizes of the pET28a, pET21a, and pBAD23 vectors are 5206 bp, 5340 bp, and 5352 bp, respectively. The specific results are shown in Fig. 3.
Fig.3 M: Marker; 1:GeDH; 2:CsTPS1; 3:GPS; 4: pET28a; 5: pET21a; 6: pBAD23
Notes: 1% Agarose gel, running time: 25min
Analysis: The obtained fragment sizes are consistent with our expectations, allowing us to preliminarily conclude that the fragments are correct.
Participants: Hanying Yu, Yilin Qian
Assembly and transformation 07/09/2025 Wednesday
Objective: The vector and target gene fragments obtained in the previous experiment were joined together to form a complete recombinant protein plasmid by Gibson cloning. Then transform the BL21 strain with plasmids carrying the target gene sequences to produce bacterial strains for subsequent protein expression and purification.
Notes: Since the three plasmids carry Kanamycin, Ampicillin, and Chloramphenicol resistance genes, the ability of the strains to grow on LB plates containing these antibiotics can be used as a preliminary indication that the three plasmids have been successfully introduced into the BL21 strains.
Results: Out of the 4 LB plates, 3 did not show any transformed colonies, and 1 was contaminated with unwanted bacteria.
Fig.4 First transformation results
Analysis: The failure may have been caused by improper handling during the transformation process. Most of the team members are not very familiar with the experimental procedures, and some details, as well as aseptic techniques, might not have been properly followed during the experiment.
Participants: Hanying Yu, Yilin Qian
Second transformation 07/12/2025 Saturday
Objective: The first transformation was unsuccessful, but one contaminated plate still showed colony growth. A second transformation was attempted, and the engineered strain was successfully constructed.
Results: One LB plate grew the desired colonies in this experiment. Whether the transformation was successful still requires further validation.
Fig.5 Second transformation results
Participants: Hanying Yu, Yilin Qian
The validation of transformation Experiment 2
Objective: Using colony PCR and gene sequencing to confirm the presence of the three recombinant protein plasmids in the engineered strains.
Design of primers for colony PCR 07/16/2025/ Wednesday
Objective: Primers were designed upstream and downstream of the target gene to perform PCR amplification on the engineered strains. The size of the bands was used to determine whether the target gene fragment was successfully assembled.
Results: Three primers were designed to perform PCR amplification on the three plasmids. The upstream and downstream regions of the primers were designed to avoid the target gene, so that if the transformation is successful, an ideal-length fragment can be amplified.
| Gene types | Forward Primer | Reverse Primer |
|---|---|---|
| GPS | 5'-CTGAATTGACTCTCTTCCG-3' | 5'-CCGGGCCGCGGACGCCAGC-3' |
| CsTPS1 | 5'-ACAGCAATGGCATCCTGGTC-3' | 5'-CTCTTCCTTTTCTCGCCACG-3' |
| GeDH | 5'-CGCTGATGTACTGACAAGCC-3' | 5'-TTAACTGCCCCGTCCTGCCG-3' |
Participants: Ethan Wu, Zibin Tang
Colony PCR 07/20/2025 Sunday
Objective: Using the three designed primers, two colonies from the second transformation plate were selected for PCR. The size of the amplified bands was observed to determine the success of the amplification.
Results: The results of DNA agarose gel electrophoresis showed that the band sizes matched the expected ones(2754bp, 3234bp and 1911bp), as shown in Fig.6. This suggests that the transformation was successfully achieved.
Fig.6 M: Marker; 1,2:GeDH; 3,4:CsTPS1; 5,6:GPS
Analysis: The results of DNA agarose gel electrophoresis are consistent with the expectations. The electrophoresis image shows a small amount of primer dimer. To present the results more clearly, only the electrophoresis image of the target band was included.
Notes: 1% Agarose gel, running time: 23min
Participants: Ethan Wu, Zibin Tang
DNA sequencing 07/21/2025 Monday
Objective: Two strains verified by colony PCR were subjected to Sanger sequencing to obtain definitive results, facilitating subsequent experiments.
Results: The sequencing results indicate that all three plasmids were successfully introduced into the BL21 strain, confirming that the engineered strain has been successfully constructed.
Analysis: The sequencing results were identical to the known sequences, indicating that the plasmid had been successfully introduced.
Participants: Ethan Wu, Zibin Tang
Bacterial Stock Preparation 07/26/2025 Friday
Objective: Prepare bacterial stocks from two colonies grown on LB plates, verified by colony PCR and sequencing, for long-term storage and use .
Results: oSix bacterial stocks were obtained and stored at -80oC.
Notes: Before bacterial stock preservation, the cultures should be incubated for a sufficient duration to ensure an adequate bacterial concentration , facilitating subsequent revival .
Participants: Zibin Tang, Yilin Qian
Validation of Growth Capacity of Engineered Strains 07/26/2025 Saturday
Objective: By tracking the OD600 during the growth of engineered E. coli, wild-type E. coli, and E. coli with empty vector, we verified whether the growth capacity of the engineered strains was affected.
Results: Take 9 x 50 mL Erlenmeyer flasks and divide them into 3 groups:
Group 1: Engineered bacteria + LB + antibiotics
Group 2: Wild-type bacteria + LB
Group 3: Empty vector bacteria + LB + antibiotics
Ensure the initial OD600 is 0.2 for each group during inoculation. Cultivate in LB medium for 24 hours. According to the analysis of the growth curve results, there was no significant difference in growth capacity among the engineered strain, the empty vector strain, and the wild-type strain. Therefore, it can be concluded that the growth capacity of the engineered strain did not significantly decrease(Fig.7).
Fig.7 Group 1: Engineered bacteria + LB + antibiotics; Group 2: Wild-type bacteria + LB;
Group 3: Empty vector bacteria + LB + antibiotics
Participants: Zibin Tang, Yilin Qian
2. Experiment Phase 2: Validation of transcription and translation.
Validation at the transcriptional level Experiment 1
Objective: At the transcriptional level, the bacteria were validated to confirm whether the target gene was successfully transcribed in BL21. The analysis of the target gene's mRNA levels provides guidance for optimizing expression conditions and ensures a solid foundation for subsequent experiments.
Bacterial Strain Activation 07/27/2025 Sunday
Objective: Revive the BL21-GPS, CsTPS1, and GeDH strains stored at -800C to restore their optimal growth conditions and accelerate proliferation.
Results: After cultivation, the bacterial strains in the shake flasks have been revived.
Participants: Zibin Tang, Yilin Qian
Scale-up cultivation 07/27/2025 Sunday
Objective: Cultivate the engineered strains in a larger culture system to obtain bacterial strains in the logarithmic growth phase.
Results: The LB liquid medium became obviously turbid, and after 6 hours of cultivation, the OD600 reached 0.698.
Analysis: When OD600 is approximately 0.6-0.8, the bacteria are in the actively growing logarithmic phase, which is suitable for inducing the transcription of the target gene .
Participants: Ruiyang Wen, Yanzhao Liu
Induction and cell harvesting 07/28/2025 Monday
Objective: Use IPTG and L-arabinose as inducers to initiate the transcription of the GPS, CsTPS1, and GeDH genes, followed by cultivation and bacterial collection.
Results: After induction, the OD600 reached 1.953. Ten milliliters of the bacterial culture were taken to collect the cells.
Participants: Ruiyang Wen, Yanzhao Liu
Total RNA extraction 07/29/2025 Tuesday
Objective: Verify whether the GPS, CsTPS1, and GeDH genes have been successfully transcribed into mRNA. Provide a template for subsequent reverse transcription and qPCR analysis.
Results: The A260/A280 ratio was 1.915 , indicating good purity and integrity with no significant DNA contamination. The concentration was 315.12 ng/µl, making it suitable for subsequent transcription-level validation experiments.
Participants: Ruiyang Wen, Yanzhao Liu
Reverse transcription to cDNA 07/29/2025 Tuesday
Objective: Reverse transcribe total RNA into cDNA to prepare for subsequent qPCR analysis.
Results: After reverse transcription, the cDNA concentration was 78.23 ng/µL, which is suitable for use as a template for qPCR.
Analysis: The theoretical concentration of cDNA is 100 ng/µL. The actual concentration is lower, likely due to losses during the experimental process.
Participants: Ruiyang Wen, Yanzhao Liu
Design of qPCR primers 07/29/2025 Tuesday
Objective: In the sequences of GPS, CsTPS1, and GeDH, select about 200 bp conserved and non-repetitive regions for amplification. Design three primers of approximately 20 bp each to prepare for the qPCR experiment.
Results: The designed primer sequences are shown in Table 5.
| cDNA types | Forward Primer | Reverse Primer |
|---|---|---|
| GPS | 5'-ATGATCTTTTCCAAAGGCCT-3' | 5'-GCGTGGACGCGGATCATCAC-3' |
| CsTPS1 | 5'-ATGAGCTCCGGTGAAACCTT-3' | 5'-CTCCTTTTCTAGCCTGACGA-3' |
| GeDH | 5'-ATGGTTCAGAACCCGGGTGC-3' | 5'-CACCTGGCAGCAGTTAGCCT-3' |
qPCR 08/01/2025 Friday
Objective: : By analyzing the data obtained from qPCR, the transcription levels of the three target genes, GPS, CsTPS1, and GeDH, were validated.
Results: We selected the bacteria before induction as the control group, while the bacteria after induction were used as the treated group. Detailed results are shown below.
| Gene types | Sample | Average of Ct values (Target genes) | Average of Ct values (Reference gene: 16S rRNA) |
|---|---|---|---|
| GPS | Treated group Control group |
22.04 26.51 |
19.56 20.02 |
| CsTPS1 | Treated group Control group |
23.09 27.53 |
19.73 20.00 |
| GeDH | Treated group Control group |
21.40 25.56 |
19.57 20.07 |
| Gene types | Sample | ??Ct | Relative expression level |
|---|---|---|---|
| GPS | Control group Treated group |
0 -4.01 |
1 16.11 |
| CsTPS1 | Control group Treated group |
0 -4.17 |
1 18.00 |
| GeDH | Control group Treated group |
0 -3.66 |
1 12.64 |
Fig.8 Relative expression level of GPS, CsTPS1 and GeDH
Analysis: From the qPCR results, we can conclude that the transcription levels of the three target genes have significantly increased after induction. Prove that all three genes can be extensively transcribed in BL21
Participants: Ethan Wu, Boyan Zhang
Validation at the Translation level Experiment 2
Objective: At the translation level, bacteria were validated to confirm whether the target gene was successfully translated into protein in BL21. This part is of great significance for the normal expression of exogenous genes in BL21.
Bacterial Strain Activation 08/02/2025 Saturday
Objective: Revive the BL21-GPS, CsTPS1, and GeDH strains stored at -80oC to restore their optimal growth conditions and accelerate proliferation.
Results: After cultivation, the bacterial strains in the shake flasks have been revived.
Participants: Yilin Qian, Ruiyang Wen
Protein Extraction and Quantification 08/04/2025 Monday
Objective: Extract total protein from the revived BL21-GPS, CsTPS1, and GeDH strains and quantify the protein concentration using the Nanodrop .
Results: Protein extraction was successful, and the concentration of all samples was adjusted to 1 µg/µL for downstream Western Blot analysis.
Participants: Yanzhao Liu, Hanying Yu
Protein Extraction and Quantification 08/04/2025 Monday
Objective: Extract total protein from the revived BL21-GPS, CsTPS1, and GeDH strains and quantify the protein concentration using the Nanodrop.
Results: Protein extraction was successful, and the concentration of all samples was adjusted to 1 µg/µL for downstream Western Blot analysis.
Participants: Yanzhao Liu, Hanying Yu
SDS-PAGE and Western Blot 08/05/2025 Tuesday
Objective: Separate the extracted proteins by SDS-PAGE and transfer them to a PVDF membrane for subsequent antibody detection of GPS, CsTPS1, and GeDH proteins.
Results: Proteins were successfully separated by SDS-PAGE, and transfer to the PVDF membrane was confirmed by Ponceau S staining.
Participants: Yanzhao Liu, Hanying Yu
Antibody Incubation and Detection 08/07/2025 Thursday
Objective:Incubate the membrane with primary and secondary antibodies to detect GPS, CsTPS1, and GeDH proteins, followed by ECL-based visualization.
Results: : Specific bands corresponding to GPS (46.9 kDa), CsTPS1 (63.7 kDa), and GeDH (41.6 kDa) were detected, as illustrated in Fig.9, confirming successful protein expression.
Ensure the initial OD600 is 0.2 for each group during inoculation. Cultivate in LB medium for 24 hours. According to the analysis of the growth curve results, there was no significant difference in growth capacity among the engineered strain, the empty vector strain, and the wild-type strain. Therefore, it can be concluded that the growth capacity of the engineered strain did not significantly decrease(Fig.7).
Fig.9 The Western blot results of GPS, CsTPS1, and GeDH.
Analysis: The SDS-PAGE results reveal only two bands , which do not align with the anticipated outcome. The lower band appears unusually thick, suggesting that due to the minimal difference in molecular weight between GPS and GeDH, the two bands have merged during electrophoresis. This observation allows us to conclude that all three proteins are successfully expressed in BL21.
Participants: Yanzhao Liu, Boyan Zhang
3. Experiment Phase 3: Analysis of products of engineered strains.
Fermentation Experiment 1
Objective: The engineered bacteria are fermented and cultured in TB medium to obtain citronellal products, and explorations are carried ou t to optimize the subsequent conditions.
The activation of the strain 08/08/2025 Friday
Objective: Restore the activity and growth and reproduction ability of the strain in a dormant state , ensure the stability of its physiological characteristics, and provide viable and consistent bacteria for subsequent fermentation production and experimental research.
Results: The activation of Escherichia coli was stopped when the OD600 value reached 0.523. At this time , the bacterial cells were in the logarithmic growth phase, with relatively good vitality and consistent physiological states, making them suitable for being transferred into the TB medium for further cultivation.
Participants: Ethan Wu, Boyan Zhang
Scale-up cultivation 07/27/2025 Sunday
Objective: Transfer the bacteria to the TB medium and conduct enlarged cultivation at 37oC and 200 rpm to obtain a larger number of bacteria, so that more proteins can be produced in the subsequent induction.
Results: ftAer culturing for 12 hours , the 1L of TB medium became obviously turbid, with distinct flocculent phenomena visible in the medium . The OD600 value reached 0.588, which is suitable for induction.
Participants: Ethan Wu, Boyan Zhang
Induction by IPTG and arabinose 08/09/2025 Saturday
Objective: Induce the BL-21-GPS-CsTPS1-GeDH strain with IPTG and arabinose at 22oC and 200 rpm to make it express proteins in large quantities .
Results: Stop the fermentation 72 hours after adding the inducer, collect the fermentation broth, and measure the OD600 value at this time, which reaches 5.213.
Participants: Yilin Qian, Yanzhao Liu
Yield analysis Experiment 2
Analyze the components of the products by HPLC 08/13/2025 Wednesday
Objective: The product components were analyzed by high-performance liquid chromatography (HPLC), and the content of citronellal in the fermentation supernatant and cell lysate was quantitatively analyzed . Meanwhile, the differences between the two were compared.
Results: Due to the limitations of laboratory conditions , we were unable to independently carry out the HPLC measurement. Therefore, we sent the samples to Beijing TD Company for analysis. Only the results are presented below.
Fig.10 The standard curve of the citronellal sample.
Fig.11 TB fermentation supernatant HPLC
Fig.12 HPLC of cell disruption solution
Fig.13 Significance Analysis of Concentration Differences between Cell Lysate and Fermentation Supernatant.
Analysis: As shown in Fig. 10 , we established a standard curve using citronellal standards with concentrations of 0.4 g/L, 0.7 g/L, 1 g/L, 1.3 g/L, and 1.6 g/L. Subsequently, we analyzed the supernatant sample of the fermentation broth, and the calculated concentration reached 0.89 g/L(Fig. 11 ). The concentration of the cell lysate was analyzed, and it reached 0.91 g/L(Fig. 12). After performing a significance analysis on the results, no significant difference was found between the two, as shown in Fig. 13. There was no significant difference between the two. To reduce the operational difficulty, the fermentation supernatant will be directly used for analysis in the follow-up.
Participants: Hanying Yu, Zibin Tang
UV absorption analysis 8/16/2025 Saturday
Objective: To verify the accuracy of the HPLC analysis results, we also used UV absorption method to detect the concentration of the fermentation supernatant . We selected the same wavelength as that in liquid chromatography to measure the absorbance, and compared the results with those of HPLC.
Results: The concentration measured by ultraviolet absorption method is basically the same as that of HPLC , so the result can be judged to be correct.
Participants: Hanying Yu, Zibin Tang
4. Experiment Phase 4: The optimization of production conditions.
Response surface design experiment Experiment 1
Objective: Use the Design-Expert 13 software to design the response surface , determine the maximum and minimum values of the experimental conditions , and provide guidance for subsequent experiments.
Response Surface Methodology (RSM) Experimental Design 08/17/2025 Sunday
Objective: Restore the activity and growth and reproduction ability of the strain in a dormant state , ensure the stability of its physiological characteristics, and provide viable and consistent bacteria for subsequent fermentation production and experimental research.
Results:
| Factors | Levels | ||
|---|---|---|---|
| Temperature(oC) | -1 | 0 | 1 |
| IPTG (mM) | 0.10 | 0.50 | 0.90 |
| l-Arabinose (% w/v) | 0.020 | 0.110 | 0.200 |
Participants: Ruiyang Wen, Yanzhao Liu
RSM Experimental 08/18/2025 Monday
Objective: Conduct experiments according to the conditions provided by Design-Expert 13 to obtain the yield of citronellal under different fermentation conditions, providing data support for the response surface.
Results:
| Std | Run | A:Temperature | B:IPTG | C:l-Arabinose | Citronellal(g/L) |
|---|---|---|---|---|---|
| 12 | 1 | 28 | 0.9 | 0.2 | 0.68 |
| 4 | 2 | 36 | 0.9 | 0.11 | 0.77 |
| 5 | 3 | 20 | 0.5 | 0.02 | 0.82 |
| 15 | 4 | 28 | 0.5 | 0.11 | 0.84 |
| 3 | 5 | 20 | 0.9 | 0.11 | 0.90 |
| 10 | 6 | 28 | 0.9 | 0.02 | 0.88 |
| 13 | 7 | 28 | 0.5 | 0.11 | 0.90 |
| 9 | 8 | 28 | 0.1 | 0.02 | 0.54 |
| 11 | 9 | 28 | 0.1 | 0.2 | 0.81 |
| 8 | 10 | 36 | 0.5 | 0.2 | 0.60 |
| 6 | 11 | 36 | 0.5 | 0.02 | 0.65 |
| 2 | 12 | 36 | 0.1 | 0.11 | 0.43 |
| 14 | 13 | 28 | 0.5 | 0.91 | 0.60 |
| 1 | 14 | 20 | 0.1 | 0.11 | 0.84 |
| 7 | 15 | 20 | 0.5 | 0.2 | 0.96 |
Multivariate quadratic response surface regression model:
Citronellal=-0.4275A2-1.18B2+0.01C2-0.145AB-0.4325AC-0.6975BC-1.18A+0.3137B+0.32C+2.84
| Source | Sum of Squares | df | Mean Square | F-value | p-value | |
|---|---|---|---|---|---|---|
| Model | 21.19 | 9 | 2.35 | 8.90 | 0.0134 | significant |
| A-Temperature | 11.16 | 1 | 11.16 | 42.18 | 0.0013 | |
| B-IPTG | 0.7875 | 1 | 0.7875 | 2.98 | 0.1451 | |
| C-l-Arabinose | 0.8192 | 1 | 0.8192 | 3.10 | 0.1388 | |
| AB | 0.0841 | 1 | 0.0841 | 0.3178 | 0.5973 | |
| AC | 0.7482 | 1 | 0.7482 | 2.83 | 0.1535 | |
| BC | 1.95 | 1 | 1.95 | 7.35 | 0.0422 | |
| A2 | 0.6748 | 1 | 0.6748 | 2.55 | 0.1712 | |
| B2 | 5.16 | 1 | 5.16 | 19.51 | 0.0069 | |
| C2 | 0.0004 | 1 | 0.0004 | 0.0014 | 0.9717 | |
| Residual | 1.32 | 5 | 0.2647 | |||
| Lack of Fit | 1.28 | 3 | 0.4272 | 11.01 | 0.0868 | not significant |
| Pure Error | 0.0416 | 2 | 0.0208 | |||
| Cor Total | 22.51 | 14 |
Analysis:We established a standard curve using citronellal standards with conFactor coding is Coded. Sum of squares is Type III - Partial. The Model F-value of 8.90 implies the model is significant. There is only a 1.34% chance that an F-value this large could occur due to noise. P-values less than 0.0500 indicate model terms are significant. In this case A, BC, B² are significant model terms. Values greater than 0.1000 indicate the model terms are not significant. If there are many insignificant model terms (not counting those required to support hierarchy), model reduction may improve your model. The Lack of Fit F-value of 11.01 implies the Lack of Fit is not significant. There is a 8.68% chance that a Lack of Fit F-value this large could occur due to noise.
Participants: Ruiyang Wen, Yanzhao Liu
RSM Analysis 08/25/2025 Monday
Objective:: Create a response surface to analyze the influence of temperature (A), IPTG (B), and l-Arabinose (C) on the yield of citronellal . The results are shown in Fig.11-13.
Results:
Fig.14 Response surface of A and B on citronellal concentration
Fig.15 Response surface of A and C on citronellal concentration
Fig.16 Response surface of B and C on citronellal concentration
Analysis: From the analysis of the response surface, contour plot, and regression equation, the optimal fermentation conditions for citronellal are as follows: a temperature of 20oC, an IPTG concentration of 0.46 mM, and an l-Arabinose concentration of 0.2% w/v. A verification experiment was conducted under the above optimized conditions.
Participants: Hanying Yu, Boyan Zhang
Analyze the components of the products by HPLC 08/26/2025 Tuesday
Objective: Analyze the product components of the fermentation supernatant after the optimization of conditions through HPLC technology, and conduct a quantitative analysis of the content of citronellal in the fermentation supernatant.
Results: We analyzed the supernatant sample of the fermentation broth after the optimization of conditions. Through calculation, the concentration of the sample reached 1.01 g/L(Fig.17).
Fig.17 Optimal fermentation conditions predicted by response surface HPLC
Analysis: From the analysis of the response surface, contour plot, and regression equation, the optimal fermentation conditions for citronellal are as follows: a temperature of 20oC, an IPTG concentration of 0.46 mM, and an l-Arabinose concentration of 0.2% w/v. A verification experiment was conducted under the above optimized conditions.
Construction of Mutant Protein 08/28/2025 Thursday
Objective: Based on the analysis of dry lab, we constructed a mutant protein（GeDH） that theoretically has the strongest binding ability to citronellol (the precursor of citronellal), and mutated four amino acids, namely Q60R, L125P, F147Y, and A300D. We hope to further increase the yield of citronellal through mutation.
Results: We analyzed the supernatant sample of the fermentation broth after the optimization of conditions. Through calculation, the concentration of the sample reached 1.01 g/L(Fig.17).
Fig.18 The conformation of the last frame in molecular dynamics simulation
In the picture, the ligand is colored blue. In 54883, the wild type is shown in blue, and the mutant is presented as sticks in dark purple. The two fit well together. We used the amino acid sequence of the protein to reconstruct the plasmid, and constructed the engineered strain using the same methods as in Experiment Phase 1 and Experiment Phase 2. The plasmids carrying GPS and CsTPS1 in the new strain are the same as the original ones, while GeDH uses the mutated sequence. Since the process is rather tedious and repetitive, the relevant procedures are not described in detail here.
Fermentation of Engineered Bacteria with Mutant Protein 09/24/2025 Wednesday
Objective: To conduct a fermentation experiment for citronellal production using the engineered strain with mutant protein, and verify whether the mutated GeDH can enhance the yield of citronellal. Due to time constraints, we commissioned Beijing TD Company to construct the engineered strain. After obtaining the engineered strain, we fermented.
Results: Culturing was carried out under the optimal culture conditions determined by response surface analysis. The results showed that the citronellal yield of the mutant strain reached 1.36 g/L(Fig.19).
Fig.19 Fermentation HPLC under optimal conditions of mutants