Difference between revisions of "Part:BBa K3182104"
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__TOC__
  
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__NOTOC__
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<span class='h3bb'><h1>Sequence and Features</h1></span>
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<partinfo>BBa_K3182108 SequenceAndFeatures</partinfo>
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<h1>Introduction</h1>
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<partinfo>BBa_K3182104 short</partinfo>
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[[File:T--Linkoping_Sweden--MechofAction.png|420px|thumb|right|<b>Figure Y.</b> Mechanism of action]]
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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 sfGFP 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.
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<br>
 
<br>
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<br>
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<h1>Introduction</h1>
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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 can also be used with methylated DNA.
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Lysk...
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<partinfo>BBa_K3182108 short</partinfo>[[File:T--Linkoping_Sweden--fusionproteinillustration.jpg|420px|thumb|right|<b>Figure 1.</b> Mechanism of action. The CBDcipA-fusion is attached to cellulose. By adding thrombin from any source the fusion protein will be cleaved and the C-terminal fusion protein will be released into the solution. By changing the fusion protein to an antimicrobial peptide/enzyme, and using the cellulose as a bandage, the peptide/enzyme can be released into a wound by native human thrombin.]]
  
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<br><br><br><br><br><br><br>
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<h3>Assembly compabilities</h3>
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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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<h2>CBDcipA and sfGFP 3D structure</h2>
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This part can be used to track purification, measure CBD binding ability and report cleavage at the thrombin site.
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[[File:T--Linkoping_Sweden--rotatingcbdanimationloop.gif|420px|thumb|left|<b>Figure Y.</b> Crystal structure of CBDcipA with a resolution of 1.75 Å which were solved by [http://www.ncbi.nlm.nih.gov/pmc/PMC452321 Tormo et al. 1989]. PDB code 1NBC. In red from the left, W118, R112, D56, H57 and Y67, thought to be the surface which interacts strongly with cellulose.]]
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[[File:T--Linkoping_Sweden--sfGFP2.png|420px|thumb|right|<b>Figure Y.</b> Crystal structure of sfGFP with a resolution of 1.4 Å which were solved by [http://www.ncbi.nlm.nih.gov/pubmed/?term=16369541 Pédelacq et al. 2006]. PDB code 2B3P. In red the chromophore can be seen. Excitation wavelength: 485 nm, emission wavelength: 510 nm]]
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<br><br><br><br><br>
  
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<h2>CBDcipA crystal structure</h2>
  
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<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br>
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[[File:T--Linkoping_Sweden--rotatingcbdanimationloop.gif|420px|thumb|left|<b>Figure 1.</b> Crystal structure of CBDcipA with a resolution of 1.75 Å which were solved by [http://www.ncbi.nlm.nih.gov/pmc/PMC452321 Tormo et al. 1989]. PDB code 1NBC. In red from the left, W118, R112, D56, H57 and Y67, thought to be the surface which interacts strongly with cellulose.]]
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<h2>Expression system</h2>
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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]). Both this part and the part were sfGFP was changed for AsPink (<partinfo>BBa_K3182000</partinfo>) showed great expression.
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<h3>Important molecular faces</h3>
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CBDcipA is composed of a nine-stranded beta sandwich with a jelly roll topology and binds a calcium ion. It further contains conserved residues exposed on the surface which map into two clear surfaces on each side of the molecule. One of faces mainly contains planar strips of aromatic and polar residues which may be the cellulose binding part. Further aspect are unknown and unique with this CBD such as the other conserved residues which are contained in a groove.  
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<h3>The choice of cellulose binding domain</h3>
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iGEM Linköping 2019 choose CBDcipA due to many other iGEM teams exploring the possibilities of this domain. Our basic design was influenced by iGEM14 Imperial, iGEM15 Edinburgh and iGEM18 Ecuador. Purification and where to place the fusion protein (N- or C-terminal) was determined by studying the former projects. CBDcipA also originates from a thermophilic bacteria which further increases the domains applications.
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<br><br><br><br><br><br><br><br>
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<h2>Expression system</h2>
  
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[[File:T--Linkoping_Sweden--expression.png|900px|thumb|center|<b>Figure B.</b> Benchling screenshot of the expression system. This expression system leads to a high expression and therefore protein yield.]]
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The part has a very strong expression with a T7-RNA-polymerase 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]). Both this part and the part were sfGFP was changed for AsPink (<partinfo>BBa_K3182000</partinfo>) showed great expression.  
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Cellulose binding domain from Clostridium thermocellum fused to Homo sapiens antimicrobial peptide LL-37. Expression of this biobrick showed no toxic effects towards the host.
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Cellulose binding domain from Clostridium thermocellum fused to Staphylococcus genus phage endolysin.
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[[File:T--Linkoping_Sweden--expression.png|900px|thumb|center|<b>Figure B.</b> Benchling screenshot of the expression system. The T7-RNA-polymerase promotor is followed by a T7 g10 leader sequence which enhances the binding to the 16S ribosomal RNA. After the leader sequence a poly A spacer is found, which has been shown to increase translation in vitro. Before the start codon a strong RBS, g10-L, followed by an AT-rich spacer can be seen, which will slightly increase translation of the following gene.]]
  
 
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===Functional Parameters===
 
===Functional Parameters===
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<partinfo>BBa_K3182104 parameters</partinfo>
 
 
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Revision as of 16:08, 6 September 2019

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
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI.rc site found at 598


Introduction

pT7-CBDcipA-sfGFP
Figure 1. Mechanism of action. The CBDcipA-fusion is attached to cellulose. By adding thrombin from any source the fusion protein will be cleaved and the C-terminal fusion protein will be released into the solution. By changing the fusion protein to an antimicrobial peptide/enzyme, and using the cellulose as a bandage, the peptide/enzyme can be released into a wound by native human thrombin.

Assembly compabilities

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 can be used to track purification, measure CBD binding ability and report cleavage at the thrombin site.






CBDcipA crystal structure

Figure 1. Crystal structure of CBDcipA with a resolution of 1.75 Å which were solved by [http://www.ncbi.nlm.nih.gov/pmc/PMC452321 Tormo et al. 1989]. PDB code 1NBC. In red from the left, W118, R112, D56, H57 and Y67, thought to be the surface which interacts strongly with cellulose.

Important molecular faces

CBDcipA is composed of a nine-stranded beta sandwich with a jelly roll topology and binds a calcium ion. It further contains conserved residues exposed on the surface which map into two clear surfaces on each side of the molecule. One of faces mainly contains planar strips of aromatic and polar residues which may be the cellulose binding part. Further aspect are unknown and unique with this CBD such as the other conserved residues which are contained in a groove.

The choice of cellulose binding domain

iGEM Linköping 2019 choose CBDcipA due to many other iGEM teams exploring the possibilities of this domain. Our basic design was influenced by iGEM14 Imperial, iGEM15 Edinburgh and iGEM18 Ecuador. Purification and where to place the fusion protein (N- or C-terminal) was determined by studying the former projects. CBDcipA also originates from a thermophilic bacteria which further increases the domains applications.










Expression system

The part has a very strong expression with a T7-RNA-polymerase 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), ([http://www.ncbi.nlm.nih.gov/pubmed/2676996 Olins et al. 1989]), ([http://www.ncbi.nlm.nih.gov/pubmed/23927491 Takahashi et al. 2013]). Both this part and the part were sfGFP was changed for AsPink (BBa_K3182000) showed great expression.

Figure B. Benchling screenshot of the expression system. The T7-RNA-polymerase promotor is followed by a T7 g10 leader sequence which enhances the binding to the 16S ribosomal RNA. After the leader sequence a poly A spacer is found, which has been shown to increase translation in vitro. Before the start codon a strong RBS, g10-L, followed by an AT-rich spacer can be seen, which will slightly increase translation of the following gene.

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]