A method for gene knockout in Gluconobacter oxidans
A technology for oxidizing glucose and acid bacteria, applied in the field of genetic engineering and bioengineering, can solve problems such as the successful development of CRISPR/Cas gene knockout system, and achieve the effect of easy operation
- Summary
- Abstract
- Description
- Claims
- Application Information
AI Technical Summary
Problems solved by technology
Method used
Image
Examples
Embodiment 1
[0038] Example 1: Shuttle plasmid p2-1-p112-Cas3-p Atse - Construction of crRNA
[0039] Primer pair F1 and R1 designed for linearization of vector p2-1,
[0040] F1:
[0041] ACCGGTTGCCGTTATTACCGGTTCGAGTGCTGG TATGTGTTCCCCGCACACGCGGGGATGAACCGTTTTTTTTGTACTGAGAGTGCACCAT (the underlined part is the homology arm sequence, the same below),
[0042] R1:
[0043] CTGGGATGCCAGCGCGCGAACAACAGTATC AATCTGCTCTGATGCCGCATAG.
[0044] The p2-1 plasmid stored in the laboratory was used as a template, and the primers were used for PCR linear amplification, and the PCR product was purified and recovered to obtain a linearized fragment p2-1 (SEQ ID NO.4).
[0045] Designed to amplify the promoter P 112 sequence of primer pair F2 and R2,
[0046] F2: GATACTGTTGTTCGCGCGC ,
[0047] R2: GGGAAATACTCCTGATTTCGTCCTG .
[0048] Using the genome sequence of Gluconobacter oxydans WSH-003 as a template, F2 and R2 were used to perform PCR amplification on it, and the PCR product was purified an...
Embodiment 2
[0061] Example 2: Construction of upstream and downstream fusion homology arms of sorbose reductase for knocking out Gluconobacter oxidans WSH-003 sorbose reductase
[0062] The primer pair F4 and R4 designed to amplify the sequence of the upstream homologous fragment of sorbose reductase,
[0063] F4: AAGGGAACGAGTCACGGAC,
[0064] R4: ATATTAACAATATAAGTGGATTAACTGCCGATAACCTCATTTTTCT .
[0065] The genome sequence of Gluconobacter oxydans WSH-003 was used as a template, and the primers were used for PCR amplification, and the PCR product was purified and recovered to obtain the upstream homologous fragment of sorbose reductase.
[0066] The primer pair F5 and R5 designed to amplify the downstream homologous fragment sequence of sorbose reductase,
[0067] F5:
[0068] AGAAAAATGAGGTTATCGGCAGTTAATCCACTTATATTGTTAATAT CGTGATTAACTAC,
[0069] R5: TCGGTCACCCCCAGGTC.
[0070] Using the genome sequence of Gluconobacter oxydans WSH-003 as a template, the primer pair F5 and R5 we...
Embodiment 3
[0072] Example 3: Knockout verification of recombinant Gluconobacter oxidans WSH-003 sorbose reductase
[0073] Will sequence the correct p2-1-p112-Cas3-p Atse -crRNA and sorbose reductase were fused with homology arms to co-transform Gluconobacter oxydans WSH-003, and after culturing on a solid sorbitol plate for about 24 hours until visible colonies grew, a single colony was randomly picked and carried out using cross-primers F4 and R5 Colony PCR verification. Colony PCR was performed using Taq PCR Master Mix, and the conditions were as follows: pre-denaturation at 94°C, 3min; amplification stage 25 cycles, 94°C, 30s, 56°C, 30s, 72°C, 2min; final extension at 72°C, 5min.
[0074] If the sorbose reductase gene is knocked out, the size of the fragment obtained by PCR is 1023bp; if the sorbose reductase gene is not knocked out, the size of the fragment obtained by PCR is 1815bp. According to the results of agarose gel electrophoresis, among the 12 single colonies picked at ra...
PUM
Login to View More Abstract
Description
Claims
Application Information
Login to View More