Notebook
Part 1 From Vanillic Acid to p-ABA
Week1 | 4.1-4.7
Validation of the hypothesis: vanillic acid and chorismic acid can undergo the same reaction to produce p-ABA. This week, we first constructed the plasmid pYB1a-pabABC.
Week2| 4.8-4.14
Calibration of the standard curve for vanillic acid and p-ABA. The standard curve for AAP was established using standard solutions of vanillic acid and p-ABA at concentrations of 0.1, 0.2, 0.3, 0.4, 0.5, and 1.
The pYB1a-pabABC plasmid was introduced into the BW25113 strain. Fermentation was conducted with the BW25113 strain into which the pYB1a-pabABC plasmid had been induced, and samples were taken at 0h and 12h to detect the production of p-ABA using High-Performance Liquid Chromatography (HPLC).
Week3 | 4.15-4.21
Bypass optimisation: Construct and introduce the R6K-dCas9(sg-nhoA) plasmid to interfere with nhoA expression. Colony PCR results of R6K-dCas9 (sg-nhoA) plasmid.
Week4 | 4.22-4.29
Conducting HPLC detection of the interference and non-interference of the nhoA group p-ABA yield. One group involved the single transformation of pYB1a-pabABC into BW25113, while another group consisted of the dual transformation of pYB1a-pabABC and R6K-dCas9(sg-nhoA) into BW25113. Samples were taken at set intervals of 0h, 6h, 12h, 18h, and 24h from 1.5mL centrifuge tubes, 10mL centrifuge tubes, and 100mL conical flasks (OD50) and OD6 to detect p-ABA yield using HPLC.
Week5 | 4.29-5.5
Bypass optimisation: Knocking out the nhoA gene. This week, we first constructed the Donor-U500D500-nhoA and pTarget-nhoA plasmids.
Week6| 5.6-5.13
Obtain the target fragment via PCR and electroporate it along with the pTarget-nhoA plasmid into the BW strain. Incubate at 37°C for 12 hours. Perform colony PCR to validate if the knockout was successful.
Positive rate 95%
Week7 | 5.14-5.21
Introduce the pYB1a-pabABC plasmid into the nhoA knockout strain BW25113 and measure the concentration of the intermediate product p-ABA at different fermentation times.
Week8 | 5.22-5.29
Optimisation of fermentation conditions:
1. Glutamine concentration gradient
2. Comparison of M9 fermentation broth and M9g fermentation broth
Week9 | 5.30-6.6
1.Glutamine concentration gradient
2.Comparison of M9 fermentation broth and M9g fermentation broth
Part 2 From p-ABA to p-AP
Week10 | 6.7-6.14
Verification of hypotheses: validating the enzymatic activity of MNX1 and optimizing the enzymatic activity efficiency of ABH60.
This week, we first constructed the plasmids pYB1a-pabABC-MNX1 and pYB1a-pabABC-ABH60.
Week11 | 6.15-6.22
We constructed plasmids pYB1a-pabABC-ABH60 and pYB1a-pabABC-MNX1 and separately introduced them into the engineered strain BW-ΔnhoA. After shaking the cultures for 12 hours, we took 1 ml of the culture and divided it into two groups: one prior to induction and one after induction. For the post-induction group, we added arabinose to induce the expression of pabABC and ABH60 genes. After incubating at 30°C for 16 hours, we took 6 OD of the culture, disrupted it by sonication, and centrifuged it at 10,000 rpm for 5 minutes. We then collected the supernatants from both the pre-induction and post-induction groups, as well as the precipitates from both groups, and added an equal volume of loading buffer for SDS-PAGE experiments.
We constructed plasmids pYB1a-pabABC-ABH60, pYB1a-pabABC-MNX1 and introduced the engineered bacteria BW-ΔnhoA respectively. After 12 hours, 1 ml bacterial suspension was divided into two groups: pre-induction group and post-induction group. After induction, Arabinose was added to induce the expression of pabABC and ABH60 genes. After 16 h at 30 °C, 6 OD bacterial suspensions were taken respectively, and centrifuged for 5 min at 10000 rpm after ultrasonic disruption. The supernatants were taken before induction, and after induction, the supernatants were precipitated before induction.
Week12|6.23-6.30
Transfer the pYB1a-pabABC-MNX1 and pYB1a-pabABC-ABH60 plasmids into BW-ΔnhoA. Sample at 12, 24, 36, and 48 hours during fermentation and use HPLC to detect the p-AP yield.
Week13|6.31-7.6
Introduce pYB1a-pabABC-ABH60 into the BW25113 engineered strains with nhoA interference and knockout, and take samples at 12h, 24h, 36h, and 48h during fermentation for HPLC detection of p-AP yield.
pYB1a-pabABC-ABH60 was introduced into BW25113 with nhoA interference and knockout, respectively. Samples were taken at 12h, 24 h, 36 h and 48 h of fermentation and p-AP production was detected by HPLC.
Part 3 From p-AP to AAP
Week14| 7.7-7.14
Verification of the hypothesis: validating the enzyme activity of NhoA controlled by the thermosensitive promoter and optimizing the enzymatic efficiency of enzyme PANAT.
We constructed an amplified pSB1c and connected it with the promoter obtained through PCR amplification, creating a vector carrying the thermosensitive promoter. We linked pSB1c-I38 with the reporter gene mCherry. We introduced the plasmid pSB1c-I38-mCherry into BW25113 and conducted three parallel experiments at 30°C and 37°C respectively. After 16 hours, we took 200μl samples and measured OD600 using a microplate reader, exciting with 552nm light and detecting the absorption at 600nm, then normalizing the absorption results using OD600.
Week15|7.15-7.22
This week, we first constructed the plasmids pSB1c-I38-nhoA and pSB1c-I38-PANAT.
We first constructed the plasmids pSB1c-I38-nhoA and pSB1c-I38-PANAT this week
Week16|7.23-7.30
We introduced pSB1c-I38-nhoA and pYB1a-pabABC-MNX1 into the nhoA knockout strain BW25113. After shaking the culture for 12 hours, pabABC-MNX1 expression was induced, and 36 hours after inducing MNX1 expression, the culture was maintained in a shaking incubator at 30℃ for a further 96 hours. Samples were taken every 12 hours starting from 12 hours after induction at 12h, 24h, 36h, 48h, and so on.
Use AAP standard products to calibrate the standard curve of AAP with concentrations of 0.2, 0.4, 0.6, 0.8, and 1.
We transferred pSB1c-I38-nhoA and pYB1a-pabABC-MNX1 into the BW-ΔnhoA. The expression of pabABC and MNX1 genes was induced after 12h of shaking, and after 36h of induction of expression of MNX1, the bacteria were placed in a 37 °C shaking table to continue cultivation for 96 h, where 12h, 24h, 36h, 48h from the beginning of induction... Samples were taken every 12 hours.
The AAP standard curve was calibrated with AAP standard at concentrations of 0.2,0.4,0.6,0.8, and 1, respectively.
At 12h, 24h, 36h, 48h after induction. The concentration of AAP at 96h.
Detect the concentration of AAP at 12h, 24h, 36h, 48h...96h after induction using HPLC.
Week17 | 7.31-8.6
After transforming pYB1a-pabABC-ABH60 and pSB1c-I38-PANAT to BW-ΔnhoA strain and inducing expression at 30℃ for 36 hours, it was incubated at 37℃ for 48 hours, with samples taken at the 48-hour mark from the start of induction. The concentration of AAP after 48 hours of induction was measured using HPLC.
Week18 | 8.7-8.14
Compare the conversion efficiencies of NhoA and PANAT enzymes.
The pYB1a-pabABC-ABH60 and pSB1c-I38-nhoA, as well as pYB1a-pabABC-ABH60 and pSB1c-I38-PANAT, plasmids were transformed into the BW-ΔnhoA. After induction at 30°C for 36 hours, the cultures were transferred to a shaker at 37°C and grown for 96 hours. Samples were taken every 12 hours from the start of induction at 12h, 24h, 36h, 48h, etc. The concentration of AAP at 12h, 24h, 36h, 48h... up to 96h post-induction was detected using HPLC.
Optimization of glycerol content in fermentation broth
Week19 | 8.15-8.22
We cotransfected pYB1a-pabABC-ABH60 and pSB1c-I38-nhoA, pYB1a-pabABC-ABH60 and pSB1c-I38-PANAT, respectively, into BW-ΔnhoA, and each was divided into two groups to ferment in fermentation broth containing 1% and 10% glycerol, respectively, all were induced at 30 °C for 36h and then changed to 37 °C for 96h. 12h, 24h, 36h, 48h... Samples were taken every 12 hours. At 12h, 24h, 36h, 48h after induction. The concentration of AAP at 96h.
Week20 | 8.23-8.30
PYB1a-pabABC-ABH60 and pSB1c-I38-PANAT were cotransferred to BW-ΔnhoA, and fermented in broth containing 1% and 10% glycerol, respectively. Samples were taken at 0h, 48h, 60h and 72h, respectively. The yields of p-AP, p-ABA and AAP in 1% and 10% glycerol groups were determined by HPLC.
Week21 | 8.31-9.6
Cotransfection of pYB1a-pabABC-ABH60 and pSB1c-I38-PANAT in BW-ΔnhoA was performed, and both were changed to 37 °C shaker for 96h after induction at 30 °C for 36h. 12h, 24h, 36h, 48h... Samples were taken every 12 hours. At 12h, 24h, 36H, 48H after induction. 96 h, the final product AAP yield was detected.
