Difference between revisions of "Part:BBa K652001"

 
 
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<partinfo>BBa_K652001 short</partinfo>
 
<partinfo>BBa_K652001 short</partinfo>
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<p><b>***Update from Americas Jamboree</b>: This RiAFP part was named best natural BioBrick at iGEM Americas Regional Jamboree!</p>
  
 
This part contains a T7 strong promoter, RBS, our Rhagium inquistor antifreeze protein (RiAFP) with a His tag at the C-terminus, followed by a terminator.  
 
This part contains a T7 strong promoter, RBS, our 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.
 +
 +
Note: although this protein does express well in E. coli, we found that we could isolate much higher yields of RiAFP by first expressing it at fusion protein with GFP, and then incubating with TEV protease to cleave the RiAFP.
 +
 +
[[Image:Western RiAFP.jpg]]
 +
 +
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
 +
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[[Image:Yale-Protein4.jpg]]
 +
 +
Figure 2: Sample image of fractions collected after His-purification of RiAFP-GFP
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[[Image:Yale-Protein5.jpg]]
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Figure 3: Figure 5: Ri-AFP-GFP (HisTrap)
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 +
[[Image:Yale-Protein6.jpg]]
 +
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Figure 4: Treatment of RiAFP-GFP fusion protein with TEV protease
 +
 +
[[Image:Yale-Protein7.jpg]]
 +
 +
Figure 5: Purified RiAFP isolated post size exclusion. Cleaved GFP-TEV and GFP-TEV-RiAFP also visible on gel.
 +
 +
[[Image:Assay1.jpg]]
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 +
Figure 6: E. coli expressing RiAFP show freeze resistance. The data are expressed as mean values (+/- standard deviatin) of three biological replicates.
 +
 +
[[Image:Assay2.jpg]]
 +
 +
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.
 +
 +
[[Image:547px-Assay3.jpg]]
 +
 +
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.
 +
 +
[[Image:532px-Assay3.jpg]]
 +
 +
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.
 +
 +
[[Image:Yale-Rats.jpg]]
 +
 +
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.
 +
 +
[[Image:Fuzzy Ball.jpg]]
 +
 +
Figure 11: Initial "fuzzy ball" crystal hit for protein crystallography, precipitant = 2.0 M ammonium carboxylate
 +
 +
[[Image:Starry Knight.jpg]]
 +
 +
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 (only RiAFP) into the genome of the EcNR2 strain using lambda red in vivo genetic engineering.
 +
 +
[[Image:lambdared colonies.jpg]]
 +
 +
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.
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<!-- Add more about the biology of this part here
 
<!-- Add more about the biology of this part here

Latest revision as of 15:21, 28 October 2011

T7-RBS-RiAFP-His-Term

***Update from Americas Jamboree: This RiAFP part was named best natural BioBrick at iGEM Americas Regional Jamboree!

This part contains a T7 strong promoter, RBS, our 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.

Note: although this protein does express well in E. coli, we found that we could isolate much higher yields of RiAFP by first expressing it at fusion protein with GFP, and then incubating with TEV protease to cleave the RiAFP.

Western RiAFP.jpg

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

Yale-Protein4.jpg

Figure 2: Sample image of fractions collected after His-purification of RiAFP-GFP

Yale-Protein5.jpg

Figure 3: Figure 5: Ri-AFP-GFP (HisTrap)

Yale-Protein6.jpg

Figure 4: Treatment of RiAFP-GFP fusion protein with TEV protease

Yale-Protein7.jpg

Figure 5: Purified RiAFP isolated post size exclusion. Cleaved GFP-TEV and GFP-TEV-RiAFP also visible on gel.

Assay1.jpg

Figure 6: E. coli expressing RiAFP show freeze resistance. The data are expressed as mean values (+/- standard deviatin) of three biological replicates.

Assay2.jpg

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.

547px-Assay3.jpg

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.

532px-Assay3.jpg

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.

Yale-Rats.jpg

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.

Fuzzy Ball.jpg

Figure 11: Initial "fuzzy ball" crystal hit for protein crystallography, precipitant = 2.0 M ammonium carboxylate

Starry Knight.jpg

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 (only RiAFP) into the genome of the EcNR2 strain using lambda red in vivo genetic engineering.

Lambdared colonies.jpg

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.




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 147
    Illegal AgeI site found at 243
    Illegal AgeI site found at 357
  • 1000
    COMPATIBLE WITH RFC[1000]