Prefix- EcoR1/Notl/Xbal - G00000 Medium Promoter - J23107 RBS - Medium/Strong - BBa_B0030 PelB - Secretion tag - J32015 - 22aa Cerastotin - Venom serine protease Fibrin Peptide - alpha - GPRP Scar - Stop Codon - Spel/Xbal mixed - G0002 RBS - Medium/Strong - BBa_B0030 DsbC - Thiol isomerase + periplasmic signal Scar - Stop Codon - Spel/Xbal mixed - G0002 B0015 - Transcription double terminator Sufix - Spel/Notl/Pstl - G00001
To obtain our goal of an accelerated clotting process, we have deployed the venom serine protease “Cerastotin” which will act on the thrombin, bypassing multiple factors in the clotting cascade and cut long fibrinogen strands into smaller fibrin “monomers” which will stop the bleeding at a greatly accelerated rate. In order for the venom to stay localized at the wound site, we have augmented a short fibrin peptide with the venom that will polymerize with the other fibrin monomers at the wound site. With the polymerization of the venom within a permanent clot and with blood pressure directing flow to the wound, the venom will not be traveling throughout the body and initiating a clot other than that of the wound site. We chose a constitutive medium strength promoter (J23107) and a medium/strong RBS for our construct in order to produce an ample amount of venom, but also allowing our Thiol isomerase time to stabilize the multiple disulphide bonds in the venom, so the protein will fold properly and its enzymatic activity increases.
To ensure our venom protein folds properly, we have a periplasmic signal attached to both the venom/fibrin protein as well as the thiol isomerase. Foreign proteins don't fold well within the rough cytoplasm, so we sent our proteins to the periplasmic space which is a more ideal situation for our venom. With addition to the DsbC, the enzymatic activity of the venom should be significantly higher which in turn should accelerate our clotting. By having our proteins in the periplasmic space, we only need to puncture the outer membrane in order to lyse the cell. This function also allows us to release our construct without introducing E.coli inside the body. As a side note, we decided to use E.Coli as our chassis because it is easier to use than other chassis such as yeast, and we have experience of working with it before. We have looked into using other chassis such as plant cells, but based on the convenience of E.Coli, we went with that.
Prefix- EcoR1/Notl/Xbal - G00000 Medium Promoter - J23107 Scar - Stop Codon - Spel/Xbal mixed - G0002 RBS - Medium/Strong - BBa_B0030 Scar - Stop Codon - Spel/Xbal mixed - G0002 Human Factor 13a - propeptide removed Thrombin Cleavage Site AntiMicrobial Peptide LL-37 Scar - Stop Codon - Spel/Xbal mixed - G0002 B0015 - Transcription double terminator Scar - Stop Codon - Spel/Xbal mixed - G0002 Sufix - Spel/Notl/Pstl - G00001
The design of our first construct induced a rapid production of clotting proteins and in order to supplement it, we have designed a construct that would stabilize the clot and sterilize the wound. In order to stabilize the clot, we added Human Factor XIIIa (propeptide removed) which is the enzyme responsible for the polymerization of fibrin monomers, by keeping the promoters and binding sites the same strength we ensure that all of the fibrin-venom strands bind at the wound site and form a permanent plug that will not be easily re-opened. Having Factor XIIIa will accelerate the rate of polymerization which further ensures that the venom-fibrin strands will stay localized.
In addition, we have added an AntiMicrobial peptide to insure that all bacteria and infection that is potentially in the wound is sterilized. We have deployed the AMP LL-37 which punctures the outer membrane of bacterial cells causing their organelles to become exposed, ending in the cells dying. We have paired the AMP with a thrombin cleavage site that when introduced to the venom serine protease Cerastotin from our original construct, will cut the AMP that is bound to Factor XIIIa at the thrombin site, and at that moment become a functional protein that sterilizes the wound.