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.

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.

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.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 894
  Illegal AgeI site found at 990
  Illegal AgeI site found at 1104
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 74
  Illegal SapI.rc site found at 87