Part:BBa_K652000
T7-RBS-eGFP-TEV-RiAFP-His-Term
This part contains a T7 strong promoter, RBS, our eGFP-fusion protein containing the Rhagium inquistor antifreeze protein (RiAFP) with a His tag at the C-terminus, followed by a terminator.
More information about this and other parts can be found on our wiki under the project section: http://2011.igem.org/Team:Yale
Cloning results were confirmed by colony PCR, restriction digest, and full sequencing.
Expression levels of up to 150mg/L of this protein are detected by UV-vis.
Figure 1: Western Blot, probed with eGFP antibody. Lane 0: ladder, Lane 1: uninduced eGFP-RiAFP in BL21, Lane 2: induced eGFP-RiAFP in BL21, Lane 3: uninduced eGFP-RiAFP in Origami, Lane 4: induced eGFP-RiAFP in Origami, Lane 5: uninduced RiAFP in BL21, Lane 6: induced RiAFP in BL21, Lane 7: uninduced RiAFP in Origami, Lane 8: induced RiAFP in Origami, Lane 9: uninduced eGFP-TEV-TmAFP, Lane 10: induced eGFP-TEV-TmAFP, Lane 11: ladder
Figure 2: Sample image of fractions collected after His-purification of RiAFP-GFP
Figure 3: Figure 5: Ri-AFP-GFP (HisTrap)
Figure 4: Treatment of RiAFP-GFP fusion protein with TEV protease
Figure 5: Purified RiAFP isolated post size exclusion. Cleaved GFP-TEV and GFP-TEV-RiAFP also visible on gel.
Figure 6: E. coli expressing RiAFP show freeze resistance. The data are expressed as mean values (+/- standard deviatin) of three biological replicates.
Figure 7: Splat assay shows concentration dependent inhibition of ice recrystallization. Ice recrystallization was allowed to take place for five hours at -10oC before imaging.
Figure 8: Splat assay shows concentration dependent inhibition of ice recrystallization. Ice recrystallization was allowed to take place for twelve hours at -10oC before imaging.
Figure 9: Capillary assay indicates that RiAFP inhibits ice recrystallization formation in a concentration-dependent manner. Recrystallization took place at -10¬oC for one hour.
Figure 10: Upon review of H&E stained sections of the liver tissue, there is an apparent difference in histological structure. Control tissue frozen with 0.9% saline and 0 mg/mL RiAFP show large noticeable patches of perforation with numerous small shrunken nuclei indicative of the early stages of cell death. Tissue frozen with 4.5 mg/mL and 9.0 mg/mL of RiAFP showed decreased perforation on the whole as well as increased cell survival and tissue integrity.
Figure 11: Initial "fuzzy ball" crystal hit for protein crystallography, precipitant = 2.0 M ammonium carboxylate
Figure 12: Initial "starry night" crystal hit, precipitant = 1.6 M sodium biphosphate, .4 M potassium phosphate
Other aspects:
We compared nematodes frozen in elution buffer, a glycerol freezing solution, freezing solution diluted in elution buffer, RiAFP (1mg/mL) added to freezing solution, and a solution of RiAFP (1mg/mL). Worms were frozen at -80oC overnight. This freezing treatment was completely lethal to the nematodes frozen in elution buffer, worms frozen in glycerol freezing solution, and worms frozen only in RiAFP solution. This large degree of lethality, especially for worms frozen in the typical freezing solution, is unusual. We suspect it is because the worms were not starved for an appropriate amount of time before freezing, or because the rate of cooling was too fast. However, interestingly, three nematodes survived after treatment with RiAFP + freezing solution. We are in the process of conducting further survivability experiments in C. elegans to better quantify any protective effects of RiAFP.
We sucessfully integrated this biobrick into the genome of the EcNR2 strain using lambda red in vivo genetic engineering.
Figure 13: sample colony plates showing sucess of lambda-red integration (top row, negative control plates with no colonies; bottom row left plate with colonies of RiAFP-GFP-Kanamycin ; bottom row right plate with colonies of RiAFP-Kanamycin. Cells grown on kanamycin
We generated over four million predicted combinatorial variants of this protein using multiplex automated genomic engineering, and are in the process of screening mutants for enhanced antifreeze properties.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 894
Illegal AgeI site found at 990
Illegal AgeI site found at 1104 - 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 74
Illegal SapI.rc site found at 87
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