Difference between revisions of "Part:BBa K5453004"
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==Induction Fermentation Protocol== | ==Induction Fermentation Protocol== | ||
| − | 1. Co-transform the plasmids R6K-dCas9-pfkA-zwf-M and pYB1c-Gatz-PGP-PGI into | + | 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. | 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. | 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. | ||
Revision as of 11:39, 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
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 1096
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 3375
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
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.