Revision as of 15:54, 23 August 2019

Introduction

Figure Y. Mechanism of action

This part consists of a cellulose binding domain (CBD) from Clostridium thermocellum cellulose scaffolding protein (CipA) with an sfGFP fused, using a flexible GS-linker (-GGGGSGGGGS-), to the CBDCipA. A thrombin cleavage site (-LVPRGS-) is added to the end of the linker and its breakage will leave a glycine and serine amino acid 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 can be used to track purification, measure CBD binding ability and report breakage of the thrombinsite.

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), ([http://www.ncbi.nlm.nih.gov/pubmed/2676996 Olins et al. 1989]), ([http://www.ncbi.nlm.nih.gov/pubmed/23927491 Takahashi et al. 2013]).

CBDcipA and sfGFP 3D structure

Figure Y. 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.

Figure Y. 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

Figure X. Basic overlook of cellulose purification.

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. Benchling screenshot of the expression system. This expression system leads to a high expression and therefore protein yield.

Usage and Biology

Figure Z Picture 1: Binding studies of the CBDcipA-sfGFP bound to bacterial cellulose. Washed three times with either 70 % ethanol, PBS or deionized water. Picture 2: Induced culture after 16 hours. E. coli BL21 (DE3) cells were grown in prescence of 25 ug/mL chlorampenicol until an OD600 of 0.8 at 37 degrees Celsius, and later induced with 0.5 mM IPTG. The induced culture were then incubated in 16 degrees Celsius for 16 hours. Picture 3: Left: CBDcipA-sfGFP bound to bacterial cellulose in form of a thin film, right: bacterial cellulose reference. Binding of CBDcipA-sfGFP was done the same way as the pictures below.

Figure A Picture 1: Lysate containing CBDcipA-sfGFP with bacterial cellulose before incubation. Picture 2: Lysate bound to bacterial cellulose after incubation in room temperature for 30 minutes on an end-to-end rotator. Picture 3: Bacterial cellulose after incubation with 70 % ethanol in room temperature for 30 minutes on an end-to-end rotator. All pictures were taken on a UV-table for better visualization of the result.

Measurement of CBD binding ability

Tracking of purification

Figure Y.

Reporter of successful cleavage and release from the cellulose binding domain

Figure Y. A kinetic experiment of thrombins protease activity. Bacterial cellulose, with CBD-sfGFP attached, were analyzed spectrophotometrically. The cellulose-CBD-sfGFP were attached to the side of wells of a 96-well plate and 200 uL 1X thrombin cleavage buffer (20 mM Tris-HCl, 150 mM NaCl and 2.5 mM CaCl2) were added. To the wells with cellulose-CBD-sfGFP and buffer, 0.03 units of human thrombin were added and fluorescence (ex. 485 nm, em. 510 nm) were measured from the bottom and up (center of the well) for 16 hours. In blue successful release of sfGFP from the CBD can be seen. In red the control experiment can be seen, where no thrombin was added.

Figure X. Visual control of human thrombin protease activity. Bacterial cellulose was incubated with CBD-sfGFP for 30 minutes on an end-to-end rotator in room temperature. After unbound protein had been removed the cellulose was washed three times with 70 % ethanol. To test the activity, 200 uL thrombin cleavage buffer (20 mM Tris-HCl, 150 mM NaCl and 2.5 mM CaCl2) were added along side 0.03 units of human thrombin to the bacterial cellulose. To the right the successful cleavage of CBD-sfGFP can be seen. The cellulose is to the left of the tube where free (cleaved at the thrombin site) sfGFP can be seen. To the left the control sample can be seen, where no sfGFP can be seen in the supernatant. The picture is taken on a 302 nm UV-table to better visualize the results.