Recombinant clostridium acetobutylicum and constructing method therefor and application of recombinant clostridium acetobutylicum
Clostridium acetobutylicum and a construction method technology, applied in the field of recombinant Clostridium acetobutylicum and its construction, improving the biofilm formation ability of Clostridium acetobutylicum, can solve the problem of toxicity, low production intensity, and raw materials High cost and other problems, to achieve the effect of improving the forming ability and butanol output
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Embodiment 1
[0042] Example 1: Construction method of knockout plasmid pNICKclos2.0-2341.
[0043] 1. Synthesis of sgRNA fragments:
[0044] In the 240bp to be deleted in the CA_C2730 gene of Clostridium acetobutylicum, three suitable targeting recognition sequences of the first 20bp of PAM were selected, and the primers sgRNA-1UP, sgRNA-2UP, sgRNA-3UP, sgRNA-DN were designed (see the table for the sequence 1). Using sgRNA-1UP / sgRNA-DN as the upstream and downstream primers and pNICKclos2.0 as the template, the sgRNA-2341-1 fragment was obtained by PCR, and its nucleotide sequence is shown in SEQ ID NO.13. Using sgRNA-2UP / sgRNA-DN as the upstream and downstream primers and pNICKclos2.0 as the template, the sgRNA-2341-2 fragment was obtained by PCR, and its nucleotide sequence is shown in SEQ ID NO.14. Using sgRNA-3UP / sgRNA-DN as the upstream and downstream primers, pNICKclos2.0 as the template, PCR to obtain the sgRNA-2341-3 fragment, its nucleotide sequence is shown in SEQ ID NO.15 ( figure ...
Embodiment 2
[0061] Example 2: Construction method of Clostridium acetobutylicum CA_C2341 gene mutant strain:
[0062] 1. The methylation of CA_C2341 knockout plasmid:
[0063] Since Clostridium acetobutylicum contains restriction endonuclease Cac824Ⅰ, which can cut unmethylated foreign DNA, it is necessary to knock out plasmid pNICKclos2.0- before transforming the knockout plasmid into Clostridium acetobutylicum. 2341 undergoes methylation modification. The specific steps are to transform the knockout plasmid pNICKclos2.0-2341 (including: pNICKclos2.0-2341-1, pNICKclos2.0-2341-2, pNICKclos2.0-2341-3) into E. coli Top10 competent ( Contains pAN2 plasmid, this plasmid has a Bacillus subtilis phage gene, can encode methyltransferase, can realize the amplification of foreign plasmid in Escherichia coli (methylation), spread to 30ug / mL erythromycin and 10ug / mL tetracycline (pAN2 plasmid has tetracycline resistance) and LB plate, cultured at 37°C for 12h, pick the transformants into liquid LB medi...
Embodiment 3
[0071] Example 3: Cultivation and fermentation method of Clostridium acetobutylicum CA_C2341 mutant.
[0072] P2 liquid medium: yeast powder 3g / L, peptone 5g / L, glucose 10g / L, ammonium acetate 2g / L, sodium chloride 3g / L, magnesium sulfate heptahydrate 3g / L, potassium dihydrogen phosphate 1g / L, Dipotassium hydrogen phosphate 1g / L, ferrous sulfate heptahydrate 0.1g / L, and the rest is water.
[0073] P2 plate medium: yeast powder 3g / L, peptone 5g / L, glucose 10g / L, ammonium acetate 2g / L, sodium chloride 3g / L, magnesium sulfate heptahydrate 3g / L, potassium dihydrogen phosphate 1g / L, Dipotassium hydrogen phosphate 1g / L, ferrous sulfate heptahydrate 0.1g / L, yeast powder 3.5g / L, the rest is water.
[0074] Fermentation medium: glucose 60g / L, ammonium acetate 2.2g / L, potassium dihydrogen phosphate 0.5g / L, dipotassium hydrogen phosphate 0.5g / L, sodium chloride 0.01g / L, magnesium sulfate heptahydrate 0.2g / L , Ferrous sulfate heptahydrate 0.01g / L, manganese sulfate monohydrate 0.01g / L, the res...
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