Difference between revisions of "Part:BBa K3081002"
 
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This part consists of araC/araBAD promoter and sfGFP fused to the C terminus of dCas9. Expression level of dCas9 induced by arabinose is tested by fluorescence of fused dCas9 linker sfGFP.
 
This part consists of araC/araBAD promoter and sfGFP fused to the C terminus of dCas9. Expression level of dCas9 induced by arabinose is tested by fluorescence of fused dCas9 linker sfGFP.
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Promoter pBAD is induced with inexpensive and non-toxic monosaccharide L-arabinose and in bacterial strains deficient in arabinose catabolism. This promoter is tightly regulated and can reach moderately high levels of downstream gene expression.In order to obtain a dynamic range and proper concentration to induce the expression of dCas9, we use cytometry to characterize the pBAD/araC promoter on respectively medium copy number pSB3C5 and low copy number pSB4A5 in TOP10 and found the fluorescense is increased as the concentration of arabinose increases (Figure 1).After we changed the downstream gene "sfGFP" with "sfGFP fused dCas9" on pSB3C5, it was observed that the expression of dCas9 from pBAD promoter can be modulated over a wide range of arabinose concentrations (Figure 2).
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<center>
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https://2019.igem.org/wiki/images/8/88/T--Peking--different_vectors_pBAD_new.png
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</center>
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<center>
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Figure 1: The response curve of pBAD dCas9-sfGFP in two vectors, respectively medium copy number pSB3C5 and low copy number pSB4A5
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</center>
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<center>
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https://2019.igem.org/wiki/images/6/60/T--Peking--different_downstream_genes-new.png
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</center>
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<center>
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Figure 2: pBAD response curve with different downstream genes
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</center>
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In order to alleviate the burden caused by dCas9,we add a degradation tag "ssrA" to the end of sfGFP. Then we found that the protein with ssrA was expressed at a lower level than the control protein, with the same concentration of inducer.
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<center>
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https://2019.igem.org/wiki/images/7/78/T--Peking--dls_ssrA.png
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</center>
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<center>
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Figure 3: Comparison between dCas9 and dCas9-ssrA system by expression level (on a low copy number plasmid pSB4A5)
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</center>
  
 
<!-- Add more about the biology of this part here
 
<!-- Add more about the biology of this part here

Latest revision as of 20:28, 21 October 2019


pBAD-dCas9-linker-sfGFP

This part consists of araC/araBAD promoter and sfGFP fused to the C terminus of dCas9. Expression level of dCas9 induced by arabinose is tested by fluorescence of fused dCas9 linker sfGFP.

Promoter pBAD is induced with inexpensive and non-toxic monosaccharide L-arabinose and in bacterial strains deficient in arabinose catabolism. This promoter is tightly regulated and can reach moderately high levels of downstream gene expression.In order to obtain a dynamic range and proper concentration to induce the expression of dCas9, we use cytometry to characterize the pBAD/araC promoter on respectively medium copy number pSB3C5 and low copy number pSB4A5 in TOP10 and found the fluorescense is increased as the concentration of arabinose increases (Figure 1).After we changed the downstream gene "sfGFP" with "sfGFP fused dCas9" on pSB3C5, it was observed that the expression of dCas9 from pBAD promoter can be modulated over a wide range of arabinose concentrations (Figure 2).

T--Peking--different_vectors_pBAD_new.png

Figure 1: The response curve of pBAD dCas9-sfGFP in two vectors, respectively medium copy number pSB3C5 and low copy number pSB4A5

T--Peking--different_downstream_genes-new.png

Figure 2: pBAD response curve with different downstream genes

In order to alleviate the burden caused by dCas9,we add a degradation tag "ssrA" to the end of sfGFP. Then we found that the protein with ssrA was expressed at a lower level than the control protein, with the same concentration of inducer.

T--Peking--dls_ssrA.png

Figure 3: Comparison between dCas9 and dCas9-ssrA system by expression level (on a low copy number plasmid pSB4A5)

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 1205
    Illegal NheI site found at 2321
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 1144
    Illegal BamHI site found at 4600
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 979
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI site found at 961
    Illegal SapI.rc site found at 5369