Plasmid

Part:BBa_K3779018

Designed by: JiaCheng, Li   Group: iGEM21_NWU-CHINA-B   (2021-06-21)


pPICZαA-SS-bgly Gene copy number

Within a certain range, the expression level of target protein increased with the increase of copy number, but when it reached a certain value, the expression level of target protein decreased with the increase of copy number. We screened for high copies by increasing the resistance concentration, and the copy number was determined by fluorescence quantification.

T--NWU-CHINA-B--_Z_A_.png

Using the previously extracted and preserved plasmid pPIC9K-SS-bgly as the template, the SS bgly gene fragment containing cleavage sites KpnⅠand NotⅠwas amplified by primers, with a size of about 1500 BP. The electrophoresis results showed that it was successfully amplified.

The following is a gel electrophoresis map of SS-bgly gene amplification.

T--NWU-CHINA-B--_SS-bgly_electrophoresis_figure_bgly_gene_amplification_.png

The two ends of the gene SS-bgly amplified by PCR were introduced into the enzyme digestion sites Kpn Ⅰ and Not Ⅰ, and the expression vector pPICZαA double enzyme digestion was performed to confirm plasmid pPICZαA-SS-bgly built successfully.

T--NWU-CHINA-B--_Colony_PCR_was_used_to_verify_positive_inverters_.png T--NWU-CHINA-B--_Identification_of_recombinant_plasmid_pPICZaA-SS-bgly_by_enzyme_digestion_.png

We compared the enzyme activities of the strains.

T--NWU-CHINA-B--_Enzyme_activity.png

The copy number of genes was determined by fluorescence quantitative PCR. Using plasmid pPIC9KSS-bgly as single copy, the copy numbers of SS-bgly gene in GS115/9K-SS-bgly 6# and GS115/9K-ZαA-SS-bgly 4# were 1.89 and 4.03, respectively, and were round 2 and 4.

T--NWU-CHINA-B--_Gene_copy_number_calculation.png

Note 2: GAP was the internal reference gene and was represented by Ref; Plasmid PPIC9K-SS-BGLY was used as reference sample and was represented by Cal. Ss-bgly stands for Target gene and is represented by Target. Strains GS115/9K-bgly 6# and GS115/9K-ZαA-bgly 4# were used as Test samples.

The expression of plasmid was optimized.

By inserting the target gene into the plasmid, we can produce the desired strain, and on the basis of which the enzyme activity efficiency can be improved.


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 2170
    Illegal XhoI site found at 271
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 2949
    Illegal NgoMIV site found at 3010
    Illegal AgeI site found at 1889
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 326
    Illegal BsaI.rc site found at 1656
    Illegal BsaI.rc site found at 2153
    Illegal BsaI.rc site found at 2360
    Illegal SapI.rc site found at 989


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