Difference between revisions of "Part:BBa K4488009"
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The construct can be cloned into an expression vector such as  pET28c in E.coli to produce a fusion protein of fuGFP with CBDcipA. The fuGFP sequence is towards the N terminus of the protein with CBDcipA (BBa_K4488024) downstream followed by a stop codon. Recognition sites for BamHI and BsaI are present before the RBS allowing golden gate cloning. XhoI and BsaI are also present downstream of the stop codon. Notably, the assembled protein is insoluble. See BBa_K3188013 for improved construct with higher fluorescence.
 
The construct can be cloned into an expression vector such as  pET28c in E.coli to produce a fusion protein of fuGFP with CBDcipA. The fuGFP sequence is towards the N terminus of the protein with CBDcipA (BBa_K4488024) downstream followed by a stop codon. Recognition sites for BamHI and BsaI are present before the RBS allowing golden gate cloning. XhoI and BsaI are also present downstream of the stop codon. Notably, the assembled protein is insoluble. See BBa_K3188013 for improved construct with higher fluorescence.
  
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===Usage and Biology===
 
===Usage and Biology===
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Our project provided preliminary evidence that CBD and cellulose can be used to purify proteins by using fuGFP-CBDcipA.
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[[File: CBD Graph 1.png|centre|thumb|700px|Figure 1: Fluorescence readings from testing fuGFP-CBD (top) and fuGFP-linker-CBD (bottom) binding to paper filter disks. 200 uL of TOP10-pUS250v3-fuGFP-CBD cell lysate was added to a 5 mm diameter paper filter disk and incubated for 1 h before washing with 200 uL NT buffer. Fluorescence measured in a plate reader shows fuGFP-CBDcipA is significantly fluorescent and causes the disk to become fluorescent. Furthermore, binding and fluorescence is greater for fusion proteins with the engineered linker.]]
  
 
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Revision as of 03:07, 11 October 2022


Fusion of free-use GFP with CBDcipA (cellulose-binding domain) at the C-terminal end

The construct can be cloned into an expression vector such as pET28c in E.coli to produce a fusion protein of fuGFP with CBDcipA. The fuGFP sequence is towards the N terminus of the protein with CBDcipA (BBa_K4488024) downstream followed by a stop codon. Recognition sites for BamHI and BsaI are present before the RBS allowing golden gate cloning. XhoI and BsaI are also present downstream of the stop codon. Notably, the assembled protein is insoluble. See BBa_K3188013 for improved construct with higher fluorescence.

Usage and Biology

Our project provided preliminary evidence that CBD and cellulose can be used to purify proteins by using fuGFP-CBDcipA.

File:CBD Graph 1.png
Figure 1: Fluorescence readings from testing fuGFP-CBD (top) and fuGFP-linker-CBD (bottom) binding to paper filter disks. 200 uL of TOP10-pUS250v3-fuGFP-CBD cell lysate was added to a 5 mm diameter paper filter disk and incubated for 1 h before washing with 200 uL NT buffer. Fluorescence measured in a plate reader shows fuGFP-CBDcipA is significantly fluorescent and causes the disk to become fluorescent. Furthermore, binding and fluorescence is greater for fusion proteins with the engineered linker.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 163
  • 1000
    COMPATIBLE WITH RFC[1000]