Difference between revisions of "Part:BBa K5036004"
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==literature characterization== | ==literature characterization== | ||
In this study,The researchers used four strains of E. coli bacteria: DH5alpha and JM109 (both dam+/dcm+), and JM110 and GM2163 (dam-/dcm-). They introduced two plasmids, pRA-cas9 and pRA-dcas9, into each strain. These plasmids express Cas9 or dCas9 proteins under the control of a powerful, constantly active promoter called PgroESL. | In this study,The researchers used four strains of E. coli bacteria: DH5alpha and JM109 (both dam+/dcm+), and JM110 and GM2163 (dam-/dcm-). They introduced two plasmids, pRA-cas9 and pRA-dcas9, into each strain. These plasmids express Cas9 or dCas9 proteins under the control of a powerful, constantly active promoter called PgroESL. | ||
+ | <html><div align="center"style="border:solid #17252A; width:100%;float:center;"><img style=" max-width:850px; | ||
+ | width:50%; | ||
+ | height:auto; | ||
+ | position: relative; | ||
+ | top: 50%; | ||
+ | left: 25%; | ||
+ | transform: translate( -50%); | ||
+ | padding-bottom:25px; | ||
+ | padding-top:25px; | ||
+ | "src="https://static.igem.wiki/teams/5036/parts/dcas9.png | ||
+ | "> | ||
+ | <p class=MsoNormal align=center style='text-align:left;border:none;width:98% ;justify-content:center;'><span | ||
+ | lang=EN style='font-size:11.0pt;line-height:115%'>(Fig.a)the presence or absence of DNA methylation (dam+/dcm+ vs. dam-/dcm-) didn't significantly affect the toxicity of Cas9 or dCas9 proteins. However, there was a slightly lower success rate in transforming dam-/dcm- strains with plasmids containing cas9/dcas9 genes compared to strains with normal methylation. This suggests that DNA methylation might play a minor role in the efficiency of plasmid transformation with these specific genes | ||
+ | </span></p></div></html> | ||
+ | |||
+ | In order to determine expression of Cas9/dCas9 in each strain of E. coli, the researcher separated the cell extracts using electrophoresis on a gel (SDS-PAGE). Finally, they visualized the proteins using two methods: staining the gel with Coomassie blue and Western blotting. | ||
+ | |||
+ | |||
+ | <html><div align="center"style="border:solid #17252A; width:100%;float:center;"><img style=" max-width:850px; | ||
+ | width:50%; | ||
+ | height:auto; | ||
+ | position: relative; | ||
+ | top: 50%; | ||
+ | left: 25%; | ||
+ | transform: translate( -50%); | ||
+ | padding-bottom:25px; | ||
+ | padding-top:25px; | ||
+ | "src="https://static.igem.wiki/teams/5036/parts/dcas9-2.png | ||
+ | "> | ||
+ | <p class=MsoNormal align=center style='text-align:left;border:none;width:98% ;justify-content:center;'><span | ||
+ | lang=EN style='font-size:11.0pt;line-height:115%'>(Fig.b&c)The experiment confirmed the production of Cas9 and dCas9 proteins in all E. coli strains containing the relevant plasmids. These proteins appeared as a band around 160 kDa on the gel and were absent in strains with empty vector controls. Interestingly, the levels of Cas9/dCas9 expression were slightly higher in strains with normal DNA methylation (dam+/dcm+) compared to those lacking methylation (dam-/dcm-). Additionally, expression levels varied between strains, with DH5alpha showing the highest and GM2163 showing the lowest. Despite lower protein expression in dam-/dcm- strains, the results suggest a possibility of slightly greater toxicity associated with Cas9/dCas9 in these strains compared to dam+/dcm+ strains. | ||
+ | |||
+ | </span></p></div></html> | ||
<!-- Add more about the biology of this part here | <!-- Add more about the biology of this part here | ||
===Usage and Biology=== | ===Usage and Biology=== |
Revision as of 09:30, 11 September 2024
d-CAS9(C)
Part Description
It is a modified version of the CRISPR-Cas9 gene editing tool which cannot cut DNA. Instead, it can bind to a specific DNA sequence guided by an RNA molecule so it can be fused to transcriptional activators or repressors. In our model dcas9 was divided into N-terminal and C-terminal fragments. So the the C-terminal fragment was grafted onto NLS to generate NLS-dCas9(C) and avoid dcas9 self assembly
Usage
This part is attached to NLS and dCas9-synRTK receptor and won’t be released until cleavage of TCS1 and TCS2 This prevents the self-assembly of the two domains of dCas9 therefore providing control over the transcription activity.
literature characterization
In this study,The researchers used four strains of E. coli bacteria: DH5alpha and JM109 (both dam+/dcm+), and JM110 and GM2163 (dam-/dcm-). They introduced two plasmids, pRA-cas9 and pRA-dcas9, into each strain. These plasmids express Cas9 or dCas9 proteins under the control of a powerful, constantly active promoter called PgroESL.
(Fig.a)the presence or absence of DNA methylation (dam+/dcm+ vs. dam-/dcm-) didn't significantly affect the toxicity of Cas9 or dCas9 proteins. However, there was a slightly lower success rate in transforming dam-/dcm- strains with plasmids containing cas9/dcas9 genes compared to strains with normal methylation. This suggests that DNA methylation might play a minor role in the efficiency of plasmid transformation with these specific genes
In order to determine expression of Cas9/dCas9 in each strain of E. coli, the researcher separated the cell extracts using electrophoresis on a gel (SDS-PAGE). Finally, they visualized the proteins using two methods: staining the gel with Coomassie blue and Western blotting.
(Fig.b&c)The experiment confirmed the production of Cas9 and dCas9 proteins in all E. coli strains containing the relevant plasmids. These proteins appeared as a band around 160 kDa on the gel and were absent in strains with empty vector controls. Interestingly, the levels of Cas9/dCas9 expression were slightly higher in strains with normal DNA methylation (dam+/dcm+) compared to those lacking methylation (dam-/dcm-). Additionally, expression levels varied between strains, with DH5alpha showing the highest and GM2163 showing the lowest. Despite lower protein expression in dam-/dcm- strains, the results suggest a possibility of slightly greater toxicity associated with Cas9/dCas9 in these strains compared to dam+/dcm+ strains.
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 577
Illegal NgoMIV site found at 650
Illegal NgoMIV site found at 1135
Illegal NgoMIV site found at 2044 - 1000COMPATIBLE WITH RFC[1000]