Difference between revisions of "Part:BBa K3081002"

Revision as of 09:13, 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).

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

Figure 2: pBAD response curve with different downstream genes

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

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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.
-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 the microplate reader 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).
+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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