Synthesis of Caffeine and 2-Phenylethyl Alcohol

The overall experimental process from objective gene to genetically engineered bacteria:

Gene purchase → plasmid construction → competent transformation → plasmid extraction → agarose gel electrophoresis → gel recovery → enzyme digestion → preparation of E.coli Nissle 1917 competence→ competent transformation → culture and fermentation → yield detection.

1. Purchase genes

The essential genes, TCS1, TcAncCS2 and TcCS2 for caffeine and aroGfbr, aro10, adh6 for 2-PE were purchased from biotech companies.

2. Construct plasmids

Plasmids were constructed by enzymatic digestion and ligation.

3. Competency transformation: Commercial E. coli DH5a was used for competent transformation and plasmid amplification.

4. Extract plasmid.

5. Enzyme validation

6. Agarose gel electrophoresis

7. Glue recovery

Note: When constructing the plasmids for 2-PE , we tried to add three genes (aro10, adh6 and aroG) on the high-copies pZE plasmid , but the construction process proved unsuccessful , probably due to the length of the plasmid.

8. Enzyme digestion

9. Preparation of E.coli Nissle 1917 COMPETENCE

10. Electroporate, and cultivate in LB

11. Culture and fermentation

[M9 medium formula: 1g/L MOPS, 2.5g/L yeast powder, 5.64g/L M9 Minimal Salt, 10g/L glucose, 1ml MgSO4·7H2O (1 M), and 50mL calcium chloride (1 M)]

Start fermentation in shake flasks. Add inducer IPTG when the OD600≈0.6-0.8, sample every 12 hours to determine OD value, and keep it for HPLC. Incubate at 37℃ for 2h, add IPTG for induction, and transfer it to a shaking table at 30℃.

12 hours to determine OD value, and keep it for HPLC. Incubate at 37℃ for 2h, add IPTG for induction, and transfer it to a shaking table at 30℃.

13. Test out (HPLC):

2-PE: 1 ‰ trifluoroacetic acid as mobile phase, acetonitrile as organic phase

CA: 2 ‰ formic acid as mobile phase, acetonitrile as organic phase

Increased fatty acid metabolism

Knock out competence gene by using CRISPR/Cas9 system's ability to edit and regulate DNA in various organisms and cell types.

Reagents:	Equipment:
. E.coli nissle 1917 . Plasmid pTarget_ competence_ N20 . Strain culture liquid	. PCR tubes . Electrophoresis apparatus . PCR instrument . 37 ℃ incubator

1、As shown in the figure, first is the schematic diagram of the principle that we use CRISPR to knock out the gene competence. Our project hopes to enhance the β- Oxidation, so we reduced the competence that inhibited the expression of the above 15 genes. N20 sequence is 5 '- GGAATACCGCTCCC-3'. This sequence will help Cas9 protein and gRNA complex locate knockout sites. When Cas9 protein finds the target sequence of PAM, it will immediately melt the base upstream of PAM and pair it with the complementary region on the guide RNA. If the complementary region and target region are correctly paired, RuvC and HNH nuclease domains will cleave target DNA after the third nucleotide base upstream of PAM.

2、We need to design two plasmids. One is pCas9, which produces Cas9 protein, and the other is pTarget, which transcribes sgRNA. Then guide it into E.coli Nissle 1917. We introduced the design plasmid, and then mixed the bacteria for shaking table culture, and kept them in the 37 ℃ incubator for 24 hours.

3、PCR experiment was used to detect the new N20 sequence in the Ptarget plasmid. The results showed that we successfully imported the N20 sequence - 5 '- GGATA ACCGCTCCCC-3'.

4、When Cas9 protein finds the target sequence of PAM, it will immediately melt the base upstream of PAM and pair it with the complementary region on the guide RNA. If the complementary region and target region are correctly paired, RuvC and HNH nuclease domains will cleave target DNA after the third nucleotide base upstream of PAM. At this time, the PCR comparison analysis is performed again to determine whether the gene knockout is successful.

5、The following figure is our design introduction plasmid diagram.

BUCT basic experimental operation process:

Reference

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