Difference between revisions of "Part:BBa K5453004"

Latest revision as of 12:00, 29 September 2024

dcas9-sgRNA-pfkA-lac operator-lacI promoter-sgRNA-zwf-lac operator-lacI promoter

dcas9-sgRNA-pfkA-lac operator-lacI promoter-sgRNA1-zwf-lac operator-lacI promoter

Sequence and Features

Design

We employed the CRISPRi technology to simultaneously inhibit the pfkA and zwf genes, introducing random mutations at the 7th and 8th nucleotide positions of their sgRNA, and constructed an R6K-dCas9-zwf-pfkA mutant library using Golden Gate technology.

Figure 1:Schematic diagram of the R6k-dcas9-pfkA-zwf-M plasmid..

Experiments

First, we amplified the sgRNA-zwf-M and sgRNA-pfkA-M fragments and purified the amplified products using a gel extraction method. Then, utilizing the Golden Gate assembly technique, which relies on type IIS restriction enzymes, we ligated the sgRNA-zwf-M and sgRNA-pfkA-M fragments into a dCas9-containing vector. After transforming the ligation products into DH5α competent cells, we performed colony PCR, restriction enzyme analysis, and sequencing validation, ultimately successfully constructing the R6K-dCas9-zwf-pfkA-M plasmid.

Figure 2:PCR Results Figure. A: Gel electrophoresis image of PCR amplification for sgRNA-pfkA-M, sgRNA-zwf-M. B: Gel electrophoresis image of colony PCR performed on 10 randomly picked colonies from the plate. C: Gel electrophoresis image of plasmid digestion with KpnI and XhoI enzymes.D: Sequencing of the correct plasmids verified by colony PCR and enzyme digestion.

Induction Fermentation Protocol

(1) Co-transform the plasmids R6K-dCas9-pfkA-zwf-M and pYB1c-Gatz-PGP-PGI into E. coli BW25113. Select 95 colonies from the validation plate and inoculate them into a 96-deep-well plate containing 800 µL of LB medium per well. Incubate at 37°C for 12 hours. (2) Transfer 8 µL of the culture into 800 µL of ZYM5052 medium, and induce expression by adding L-arabinose and IPTG at appropriate concentrations. Incubate the cultures at 30°C for 18 hours, and subsequently measure the OD600 values. (3)Normalize the cell density by transferring an equal amount of biomass into 200 µL of M9 medium supplemented with 20 g/L glucose, and ferment at 30°C for 12 hours.

Concentration Measurement

(1) Collect samples from the shaker and centrifuge at 13,000 rpm for 10 minutes. (2) Dilute the samples 5-fold by adding 100 µL of the sample to 400 µL of distilled water. (3)Mix the solution thoroughly. (4)Add 500 µL of resorcinol solution (0.75 g/mL) to the diluted sample. (5)Incubate the mixture at 100°C for 20 minutes. (6) Cool the samples at 4°C for 5 minutes. (7)Transfer 200 µL of the cooled mixture to a 96-well plate. (8)Measure absorbance at 405 nm using a microplate reader. (9)Record and analyze the results. This version refines clarity and enhances the technical tone to sound more professional.

Figure 3:The fluorescence value detected by the Microplate reader.

Figure 4:The blue bar indicates the yield of Tagatose; the yellow circle indicates OD600

Short Summary

Figure 5: The sequencing results of the mutations at the 7、8bp position in sgRNA7 of DT-D9.

A good mutant was identified and named DT-D9. Compared to the non-inhibited strain DT1, the tagatose yield of DT-D9 increased by 3.34-fold, reaching a D-tagatose yield of 634.66 mg/L.Through sequencing the mutant, we found that the 7th and 8th base pairs of sgRNA-pfkA mutated from GG to TG, and the 7th and 8th base pairs of sgRNA-zwf mutated from AT to CT. By altering the 7th and 8th base pairs of the sgRNA, the binding efficiency of dcas9 to the target gene can be modified, thereby achieving varying degrees of inhibition. The best mutant, DT-D9, showed good growth and higher yield, proving that our strategy was effective.