Team:Kyoto/Proof
Contents
Concept
Therapeutics that directly approach NoV itself had not yet been established. We therefore aimed to develop therapeutic application for E. coli by binding them to NoV-like particles (NoVLP, only the capsid proteins of NoV) and swiftly removing them from human digestive system by binding them also to cellulose. For such biodevice to function, surface display systems, INPNC and BclA, plays an indispensable part. We first enhanced upon these existing BioBrick parts, confirmed their functionality and used them for the surface expression of anti-NoV scFv and CBDcex, examining its binding to NoVLP and cellulose, respectively.
Proof of concept
Enhancement of existing BioBrick parts
We constructed INP-His, BclA-His encoding plasmids. INPNC/BclA are already registered BioBrick parts, and we added His-tag to them. This addition introduces two new functions to the fusion protein:
1A) First, we can use anti-His tag antibody to detect their surface expression regardless of passenger protein placed downstream. We confirmed this with detections of the INPNC-His- and BclA-His-(passenger protein) by Western blot using anti-His tag antibody. (Fig1)
Marker, Negative Control (BBa_K165002), BclA-His-scFv (33kDa), INPNC-His-scFv (63kDa), BclA-His-CBDcex (17-kDa), INPNC-His-CBDcex (47-kDa), Positive Control (BBa_K875004)(43-kDa)
Whole-cell lysates were prepared from E. coli DH5alpha strain carrying various plasmids shown in the figure. Samples were separated by 10% SDS-PAGE and transferred to a PVDF membrane. His-tagged proteins were visualized by anti-His tag monoclonal antibody (clone #OGHis, MBL, Japan).
1B) Second, INPNC-His- and BclA-His-(passenger protein) can be purified using His tag’s affinity to nickel columns even in denaturing conditions to recollect it from membrane fractions of E.coli. This would allow fusion protein detection even if the protein’s expression levels are low, further strengthening the 1A’s function. We tested this function with Western blot. Sample was prepared by obtaining the membrane fraction from the E. coli lysate by ultracentrifugation, and solubilized them using 7M guanidine-HCl, then purification of the target protein using Nickel Sepharose. (Fig 2)
Membrane fraction was solubilized and used for Nickel Sepharose purification and precipitates were examined by Western blotting against His-tag. The band corresponding with INPNC-His-scFv (63kDa) INPNC-His-CBDcex (47kDa) was observed in their respective lanes, while the band corresponding with BclA could not be observed. Marker, Negative Control (BBa_K165002), BclA-His- CBDcex (17kDa), INPNC-His-CBDcex（47kDa）, BclA-His-scFv (33kDa), INPNC-His-scFv (63kDa), Positive Control (BBa_K875004)(43kDa)
These two results confirms the enhanced functions of INPNC and BclA parts existing in the iGEM Regisrtry.
Observation of E.coli’s binding to NoVLP
Using surface expressed scFv, we tested E. coli’s binding to NoVLP. First, we constructed INPNC-His-scFv and BclA-His-scFv encoding plasmid. This plasmid satisfies criteria for BioBrick parts. (LINK) We transformed E. coli (strain:DH5α)with this plasmid, and observed its interaction with NoVLP. (Fig3) We observed NoVLP only on the surface of INPNC-His-scFv expressing E. coli, as NoVLP could not be observed in E.coli expressing BclA-His-scFv, (Which may be due to low expression levels, seen in Fig 3), this NoVLP is not unspecifically mixed with the sample. Thus we concluded that we observed the binding of our INPNC-His-scFv expressing E. coli to NoVLP. Visit our description (LINK) and materials & methods (LINK) for more information on the figure and protocols used.
1, 2. E. coli expressing BclA-His-scFv. 3-6. E. coli expressing INPNC-His-scFv, 7, 8. Sample with only NoVLP. 2-microm scale bars are shown in each picture.
We have thus proved our BioBrick parts, INPNC-His-scFv, to bind to NoVLP as it was designed to do.
Observation of E.coli’ s binding to cellulose
Using surface expressed CBDcex, we tested our other concept, which is the binding of E. coli to cellulose. First, we constructed a plasmid encoding INPNC-His-CBDcex. This plasmid satisfies criteria for BioBrick parts. (LINK) We transformed E. coli using this plasmid, and examined its binding to cellulose powder through fluorescence microscopy. Far more E. coli expressing INPNC-His-CBDcex were observed in the periphery of the cellulose particles compared to negative control E. coli without CBD, and thus we concluded that INPNC-His-CBDcex expressing E. coli have bound to cellulose powder. See description page (LINK) for more detail
