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Team:Utah State/Results
From 2013.igem.org
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Text descriptions go here.
To demonstrate that the N-terminal 10x His Tag (BBa_K1162009) functions correctly, it was cloned in front of Green Florescent Protein (GFP)with the lac promoter+rbs system (BBa_K208010)to give the complete construct BBa_K1162013. This construct was expressed in E. coli grown in 50mL LB media with the addition of chloramphenicol and induced with IPTG. After allowing to grow overnight the cells were spun down and protein was purified with a nickel spin column (see protein purification protocol on protocols page). Fractions from this nickel spin column procedure were saved and run on an SDS-PAGE gel (see below). Since this was a GFP purification procedure, the different fractions were dotted on parafilm and place on a UV gel box to visualize the protein (see below).
From the SDS-PAGE gel it can be seen that there is pure GFP (~26.9 kDa) in the elution fraction number 2 and 3. The wash fractions do not appear to have any GFP which indicates that the N terminal 10x His Tag is strongly bound to the Nickel Column during washing steps. Coupled with the GFP dot image it is clear that this method of purification works as desired and adds another purification system to the registry.
After demonstration that the N-terminal purification system functioned as desired, other constructs were built to purify protein using this method.
To demonstrate that antimicrobial spider silk could be manufactured in E. coli, the AMP LL-37 (BBa_K1162006) was fused to 8 repeats of spider silk (BBa_K844004. A lac inducible promoter system (BBa_K208010), 10x His Tag (BBa_K844000), and double terminator (BBa_B0015) were added to create the first antimicrobial spider silk generator with BioBricks.
To visually monitor the structures of AMPs, amino acid sequences were entered into the Protein Homology/analogY Recognition Engine (PHYRE2,Protein structure prediction on the web: a case study using the Phyre server Kelley LA and Sternberg MJE. Nature Protocols 4, 363 - 371 (2009),http://www.sbg.bio.ic.ac.uk/~phyre2/html/page.cgi?id=index). Output for each of the AMPs used by Utah State iGEM 2013 team is given below. Note: Grammistin (BBa_K1162003) was too small to model with this program. Interestingly when amino acid sequence for each AMP was entered into the program fused with 10x Histag, no change was seen the in the protein structure.
EcAMP (BBa_K1162001) structure
Spheniscin (BBa_K1162002) structure
Cg-Defh1 (BBa_K1162004)structure
Scygonadin (BBa_K1162005)structure
LL37(BBa_K1162006)structure
WAM1(BBa_K1162007)structure
OHCATH (BBa_K1162008)
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OptKnock, a program that optimizes the flux through an objective reaction by systematically searching for reactions to “knock out” (by setting the upper and lower bounds of those reactions to zero) was performed for Grammistin(BBa_K1162003), LL37 (BBa_K1162006), and WAM1(BBa_K1162007).
Production envelope for Grammistin containing 3 knockouts
From carrying out OptKnock it was found that it was not able to find an optimal/maximal production rate for Grammistin(maximum production rate= 2.83 E-10 or zero). The other AMPs analyzed (LL37 and WAM) yielded similar results to Grammistin and hence additional AMPs were not modeled using OptKnock as each AMP took many hours to analyze.
