Difference between revisions of "Part:BBa K3182000"

 
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<partinfo>BBa_K3182000 short</partinfo>
 
<partinfo>BBa_K3182000 short</partinfo>
  
A cellulose binding domain (CBDcipA) from Clostridium thermocellum Cellulose scaffolding protein (CipA) which can be used to purify or attach proteins to cellulose, this part has a AsPink fused to the CBDcipA. The part also has a flexible GS-linker (-GGGGSGGGGS-) with a thrombin site (-LVPRGS-, thrombin RS) added at the end, clevage with thrombin will add one glycine and one serine to the N-terminal of the C-terminal fusion protein of the CBDcipA. Also added a BamHI recognition sequence (BamHI RS) to enable changeable fusion protein to the CBDcipA. BamHI was chosen because its RS codes for one glycine and one serine, fitting it to the end of the thrombin site, BamHI is also a cheap enzyme and can be used with methylated DNA.
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This part consists of a cellulose binding domain (CBD) from Clostridium thermocellum cellulose scaffolding protein (CipA) with an AsPink-chromoprotein fused,  using a flexible GS-linker (-GGGGSGGGGS-), to the CBDCipA. A thrombin cleavage site (-LVPRGS-) is added to the end of the linker. Breakage of the site will leave a glycine and serine aminoacid attached to the N-terminal of the AsPink fusion protein.  
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An internal BamHI recognition sequence (RS) has been added to enable changeable fusion proteins. BamHI was chosen because its RS codes for glycine and serine, fitting it to the end of the thrombin site. It is also cost-effective enzyme and is unaffected by methylated DNA.
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This part uses an expression system with a T7 promotor (<partinfo>BBa_I719005</partinfo>) as well as a 5'-UTR (<partinfo>BBa_K1758100</partinfo>) region which has been shown to further increase expression in E. coli (<partinfo>BBa_K1758106</partinfo>), ([http://www.ncbi.nlm.nih.gov/pubmed/2676996 Olins et al. 1989]), ([http://www.ncbi.nlm.nih.gov/pubmed/23927491 Takahashi et al. 2013]).  
  
The part has a very strong expression with a T7 promotor (<partinfo>BBa_I719005</partinfo>) as well as a 5'-UTR (<partinfo>BBa_K1758100</partinfo>) region which has been shown to further increase expression in E. coli (<partinfo>BBa_K1758106</partinfo>), ([http://www.ncbi.nlm.nih.gov/pubmed/2676996 Olins et al. 1989]), ([http://www.ncbi.nlm.nih.gov/pubmed/23927491 Takahashi et al. 2013]).
 
  
 
[[File:T--Linkoping_Sweden--CBD-AsPink.jpeg|430px|thumb|center|<b>Figure 1.</b> E. coli BL21 cells expressing this biobrick, incubated for 16 hours at 16°C at 80 rpm in 1 litre of LB-miller.]]
 
[[File:T--Linkoping_Sweden--CBD-AsPink.jpeg|430px|thumb|center|<b>Figure 1.</b> E. coli BL21 cells expressing this biobrick, incubated for 16 hours at 16°C at 80 rpm in 1 litre of LB-miller.]]

Latest revision as of 18:56, 13 August 2019


pT7-CBDcipA-AsPink

This part consists of a cellulose binding domain (CBD) from Clostridium thermocellum cellulose scaffolding protein (CipA) with an AsPink-chromoprotein fused, using a flexible GS-linker (-GGGGSGGGGS-), to the CBDCipA. A thrombin cleavage site (-LVPRGS-) is added to the end of the linker. Breakage of the site will leave a glycine and serine aminoacid attached to the N-terminal of the AsPink fusion protein.

An internal BamHI recognition sequence (RS) has been added to enable changeable fusion proteins. BamHI was chosen because its RS codes for glycine and serine, fitting it to the end of the thrombin site. It is also cost-effective enzyme and is unaffected by methylated DNA.

This part uses an expression system with a T7 promotor (BBa_I719005) as well as a 5'-UTR (BBa_K1758100) region which has been shown to further increase expression in E. coli (BBa_K1758106), (Olins et al. 1989), (Takahashi et al. 2013).


Figure 1. E. coli BL21 cells expressing this biobrick, incubated for 16 hours at 16°C at 80 rpm in 1 litre of LB-miller.
Figure 2. Lysated (via sonication) BL21s which are expressing the biobrick. It is lysate from the culture above in figure 1. Incubated for 16 hours at 16°C at 80 rpm in 1 litre of LB-miller.
Figure 3. Centrifuged lysate of BL21 culture which express the biobrick. It is the same culture as figure 1 and 2. A pellet of non-lysated bacteria can be observed.




































Potential Usages

AsPink can for example be used as a reporter for CBD binding ability, track purification of the CBD or report linker breakage. By including a BamHI site on the linker; the asPink can be switched with another protein.

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 580
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]