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]

Introduction

pT7-CBDcipA-PlyF307-SQ8C This part consists of a cellulose binding domain (CBD) from Clostridium 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-fusion 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 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.

Acineto phage muramidase

Fused to the CBD in this part is an endolysin from the Acineto phage, PlyF307, which is classified as a cell-wall hydrolase. Fused to the endolysin is an antimicrobial peptide from the hepatitis B virus. The version of this protein were inspired by [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4862495/ Thandar et al. 2016]. The endolysin is designed to battle the Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, Enterobacter spp. family of pathogens (ESKAPE). ESKAPE is a family(ies) of bacteria which has multiple understrains that has evolved resistance to the most commonly used antibiotics.

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. 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.

Usage and Biology

Fitness cost of expression for PlyF307-muta1

Figure B.

Antimicrobial activity of PlyF307-muta1