Part:BBa_K3182100

Introduction

pT7-CBDcipA-pCons-AsPink

Figure Y. Mechanism of action. The CBDcipA-sfGFP is attached to cellulose. By adding thrombin from any source the fusion protein will be cleaved and sfGFP 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.

This part consists of a cellulose binding domain (CBD) from Clostridium thermocellum (C. thermocellum) cellulose scaffolding protein (CipA) and is a central part Clostridium thermocellum's cellusome. The CBD was fused to sfGFP in this part to easily track the binding capacities and to test our release mechanism. The CBD-sfGFP were fused using a flexible GS-linker (-GGGGSGGGGS-). A thrombin cleavage site (-LVPRGS-) was added to the end of the linker and its breakage will leave a glycine and serine attached to the N-terminal of the sfGFP fusion protein.

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.

For usage and information of CBDcipA please see: BBa_K3182000 and BBa_K3182108.

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.

Theoretical usage of this method

This part utilizes a pCons-AsPink dropout enabling colour-screening for positive colonies. Using BamHI and PstI or SpeI on both this part assembled in pSB1C3 (or vector of choice) and the insert of choice will yield a fusion protein between CBDcipA and the insert. The fusion protein can later be cleaved with thrombin to yield two separate proteins. The C-terminal fusion will have one glycine and one serine added to the N-terminal of the protein.

Figure 1. The first plasmid (left) contains pCons-AsPink which results in pink colonies. When BamHI is used together with either SpeI or PstI pCons-AsPink is cut out (middle plasmid) and replaced with a compatible biobrick such as an antimicrobial agent (right plasmid). The colonies will then be white due to pCons-AsPink being cleaved from the plasmid. This results in a "pink-white screening".

Examples of biobricks assembled with this method

This method was used to assemble: BBa_K3182103, BBa_K3182104, BBa_K3182105, BBa_K3182106, BBa_K3182107, BBa_K3182108.

Usage and Biology

Design any gene which you want to be fused to the CBDcipA with a BamHI recognition sequence in the 5'-end. The biobrick suffix can be used in the 3'-end. Cut the vector and insert with BamHI and PstI (SpeI also works), remove enzymes and mix, no need for gel purification. Using very high molar ratios might not yield any pink colonies at all, a molar ratio insert to vector of 7-20:1 will yield some pink colonies. Transform the host (BL21 (DE3) for quickest results) and incubate at 37 degrees Celsius overnight, if the color is weak or can't be seen, incubate in 24-37 degrees for an additional 16-24 hours.

Figure 2. E. coli (BL21) cells used for pink-white screening, the cells were incubated for 16 hours in 37 degrees Celsius. BBa_K3182100 was cut with BamHI and PstI to remove pCons-AsPink and BBa_K3182006 (magainin 2) and BBa_K3182104 (CHAP) was ligated into the plasmid. The white colonies indicate a successful ligation. All the colonies that were later colony screened with PCR amplification of the insert and the ampified strand was run on an agarose gel. The gel implied that all screened colonies was successful, i.e. contained BBa_K3182100 with magainin 2 / CHAP instead of pCons-AsPink.

INSERT AGAROSE GEL HERE