Part CollectionThis part collection serves as a complete tool for assembly, expression and immobilization of many antimicrobial agents. The basic part, BBa_K3182001, is one of the central parts of the project. This part would bind to polysaccaride materials and be immobilized on them. To utilize this domain we had to figure out a way to assemble multiple fusion proteins.
Immobilization on polysaccaride materials and aseemblyImmobilization was achieved using our method "pink-white screening (BBa_K3182100)" where the chromoprotein AsPink (BBa_K1033933) was removed and instead replaced with one fusion protein bound to the carbohydrate binding domain (CBD). By having a constitutive promotor, (BBa_J23100) upstream of AsPink, the protein would always be expressed which could be seen as pink colonies. When removing this promotor and AsPink the pink color would disappear. This in turn made the synthesis and ligation much easier and faster. In total, this method was used 13 times.
Charaterization of the CBDThe fusion protein CBD-sfGFP (BBa_K3182100) was instead used for characterization of the CBD. This fusion protein turned out to be very easy to express and the intensity of this protein was well above what iGEM19 Linköping needed. Binding CBD-sfGFP to cellulose and other materials yielded enough fluorescence to be seen with the naked eye in white light.
Antimicrobial agentsAfter the CBD had been characterized according to our needs, the antimicrobial agents (BBa_K3182103, BBa_K3182104, BBa_K3182107) which iGEM19 Linköping chose could be immobilized on cellulose. And as we quickly noticed, the expression of these agents was possible through utilizing the CBD as a deactivation domain. The antimicrobial agents also showed great affect on bacteria of both the gram negative and the gram positive type.
ConclusionsAll these biobricks together created a small library of antimicrobial agents which could be efficiently expressed, purified with the CBD, immobilized on different polysaccaride materials and cleaved with thrombin to release the agents deep into wounds. The team from Linköping 2019 hope that this product can be utilized as a cheaper alternative to other bandages and antibiotics currently in use. Hopefully it will help a lot of patients suffering from burns and other skin wounds, in addition to preventing antibiotic resistance.
Table 1. The parts which were central to the project, from start to finish.
|Biobrick ID||Short name||Function||Size||Designed by|
|BBa_K3182001||CBDcipA||Carbohydrate binding domain with a C-terminal linker||516 bp||Oliver Hild Walett, Johan Larsson|
|BBa_K3182100||pT7-CBDcipA-pCons-AsPink||Carbohydrate binding domain assembly vector||1334 bp||Oliver Hild Walett|
|BBa_K3182108||pT7-CBDcipA-sfGFP||Characterization of the carbohydrate binding domain||1305 bp||Oliver Hild Walett|
|BBa_K3182103||pT7-CBDcipA-PlyF307-SQ8C||CBD fusion - Acinetobacter genus lysin||1029 bp||Oliver Hild Walett|
|BBa_K3182104||pT7-CBDcipA-CHAP(K)||CBD fusion - Staphylococcus genus lysin||1074 bp||Oliver Hild Walett, Leo Juhlin|
|BBa_K3182107||pT7-CBDcipA-Pln1||CBD fusion - Antibacterial peptide Pln1||702 bp||Oliver Hild Walett|