Difference between revisions of "Part:BBa K4808003"

 
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rhtA gene encodes a strong threonine exporter and transfers threonine out of the cell.  The gene rhtA knockout resulted in the reduction of extracellular secretion of threonine so there are more amount of threonine can be kept inside the cell for the further production of a-KB.  We design the pTarget plasmid that carrying specific gRNA sequence which can identity the rhtA gene, then we obtained donorDNA through two rounds of PCR. The donorDNA was used for homologous recombination with genomic DNA. We then put this pTarget plasmid and donor DNA  into AIS-0 strains that has already carried pEcCas plasmid for the CRISPR-CAS 9 knockout experiment. (We referred to the experimental procedures published by Qi Li, Bingbing Sun, et al. in 2020) Through the results of colony PCR and gene sequencing, we confirmed the successful knockout of rhtA.
 
rhtA gene encodes a strong threonine exporter and transfers threonine out of the cell.  The gene rhtA knockout resulted in the reduction of extracellular secretion of threonine so there are more amount of threonine can be kept inside the cell for the further production of a-KB.  We design the pTarget plasmid that carrying specific gRNA sequence which can identity the rhtA gene, then we obtained donorDNA through two rounds of PCR. The donorDNA was used for homologous recombination with genomic DNA. We then put this pTarget plasmid and donor DNA  into AIS-0 strains that has already carried pEcCas plasmid for the CRISPR-CAS 9 knockout experiment. (We referred to the experimental procedures published by Qi Li, Bingbing Sun, et al. in 2020) Through the results of colony PCR and gene sequencing, we confirmed the successful knockout of rhtA.
  
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===Usage and Biology===
 
  
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    <h2><b>Characterization</b></h2>
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<span class='h3bb'>Sequence and Features</span>
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    <p>rhtA gene encodes a strong threonine exporter and transfers threonine out of the cell. The gene rhtA knockout
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<partinfo>BBa_K4808003 SequenceAndFeatures</partinfo>
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        resulted in the reduction of extracellular secretion of threonine so there are more amount of threonine can be
 +
        kept inside the cell for the further production of a-KB. We design the pTarget plasmid that carrying specific
 +
        gRNA sequence which can identity the rhtA gene, then we obtained donorDNA through two rounds of PCR. The
 +
        donorDNA was used for homologous recombination with genomic DNA. We then put this pTarget plasmid and donor DNA
 +
        into AIS-0 strains that has already carried pEcCas plasmid for the CRISPR-CAS 9 knockout experiment. (We
 +
        referred to the experimental procedures published by Qi Li, Bingbing Sun, et al. in 2020) Through the results of
 +
        colony PCR and gene sequencing, we confirmed the successful knockout of rhtA. </p>
 +
src=https://static.igem.wiki/teams/4808/wiki/parts1.png
 +
    <p>Figure 1: （A）the design of pEcCas、pTarget plasmid and donorDNA for gene knockout (B) verified the construction of
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        pTarget-g-rhtA result through the sequencing testing. (C) colony PCR to respectively determine the knock-out of
 +
        rhtA (D) verified the knock-out result through the sequencing testing </p>
  
  

Revision as of 10:33, 10 October 2023


g-rhtA

rhtA gene encodes a strong threonine exporter and transfers threonine out of the cell. The gene rhtA knockout resulted in the reduction of extracellular secretion of threonine so there are more amount of threonine can be kept inside the cell for the further production of a-KB. We design the pTarget plasmid that carrying specific gRNA sequence which can identity the rhtA gene, then we obtained donorDNA through two rounds of PCR. The donorDNA was used for homologous recombination with genomic DNA. We then put this pTarget plasmid and donor DNA into AIS-0 strains that has already carried pEcCas plasmid for the CRISPR-CAS 9 knockout experiment. (We referred to the experimental procedures published by Qi Li, Bingbing Sun, et al. in 2020) Through the results of colony PCR and gene sequencing, we confirmed the successful knockout of rhtA.


Characterization

rhtA gene encodes a strong threonine exporter and transfers threonine out of the cell. The gene rhtA knockout resulted in the reduction of extracellular secretion of threonine so there are more amount of threonine can be kept inside the cell for the further production of a-KB. We design the pTarget plasmid that carrying specific gRNA sequence which can identity the rhtA gene, then we obtained donorDNA through two rounds of PCR. The donorDNA was used for homologous recombination with genomic DNA. We then put this pTarget plasmid and donor DNA into AIS-0 strains that has already carried pEcCas plasmid for the CRISPR-CAS 9 knockout experiment. (We referred to the experimental procedures published by Qi Li, Bingbing Sun, et al. in 2020) Through the results of colony PCR and gene sequencing, we confirmed the successful knockout of rhtA. 

src=parts1.png

Figure 1: （A）the design of pEcCas、pTarget plasmid and donorDNA for gene knockout (B) verified the construction of pTarget-g-rhtA result through the sequencing testing. (C) colony PCR to respectively determine the knock-out of rhtA (D) verified the knock-out result through the sequencing testing