Difference between revisions of "Part:BBa K3182107"

Revision as of 09:24, 1 August 2019

pT7-CBDcipA-Pln1

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. 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. This part has a Acinetobacter phage endolysin fused to the CBDcipA.

Pln1...

CBDcipA and Acinetobacter phage muramidase crystal structure

Figure X. 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, W118, R112, D56, H57 and Y67, thought to be the surface which interacts strongly with cellulose.

Figure Y. Crystal structure of PlyF307 / Acinetobacter phage muramidase with a resolution of 1.2 Å which were solved by [http://www.ncbi.nlm.nih.gov/pubmed/?term=29882827 Sykilinda et al. 2018]. PDB code 6ET6. In red the C-terminal alpha-helix can be seen, which has a proposed role of activating the muramidase against live bacterial cells from inside or outside of the cells at critical concentration.

Expression system

The part has a very strong expression 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), ([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.

Cellulose binding domain from Clostridium thermocellum fused to Lactobacillus plantarum antibacterial peptide. Designed to target bacteria. Expression of this biobrick showed no toxic effects towards the host.

Usage and Biology

Figure x

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
COMPATIBLE WITH RFC[12]
21
INCOMPATIBLE WITH RFC[21]
Illegal BglII site found at 653
Illegal BamHI site found at 580
23
COMPATIBLE WITH RFC[23]
25
COMPATIBLE WITH RFC[25]
1000
COMPATIBLE WITH RFC[1000]

@@ Line 3: / Line 3: @@
 <partinfo>BBa_K3182107 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 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. 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. This part has a Acinetobacter phage endolysin fused to the CBDcipA.
+Pln1...
+<h2>CBDcipA and Acinetobacter phage muramidase crystal structure</h2>
+[[File:T--Linkoping_Sweden--rotatingcbdanimationloop.gif|420px|thumb|left|<b>Figure X.</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, W118, R112, D56, H57 and Y67, thought to be the surface which interacts strongly with cellulose.]]
+[[File:T--Linkoping Sweden--plyFcrystal.png|420px|thumb|right|<b>Figure Y.</b> Crystal structure of PlyF307 / Acinetobacter phage muramidase with a resolution of 1.2 Å which were solved by [http://www.ncbi.nlm.nih.gov/pubmed/?term=29882827 Sykilinda et al. 2018]. PDB code 6ET6. In red the C-terminal alpha-helix can be seen, which has a proposed role of activating the muramidase against live bacterial cells from inside or outside of the cells at critical concentration.]]
+<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br>
+<h2>Expression system</h2>
+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.
+[[File:T--Linkoping_Sweden--expression.png|900px|thumb|center|<b>Figure B.</b>]]
 Cellulose binding domain from Clostridium thermocellum fused to Lactobacillus plantarum antibacterial peptide. Designed to target bacteria. Expression of this biobrick showed no toxic effects towards the host.