Difference between revisions of "Part:BBa K4429008"
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Results: The cloning was successful.
 
Results: The cloning was successful.
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[[image:BBa_K4429008_0.png|500px]]
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[[image:BBa_K4429008_1.png|500px]]
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<b> PURIFICATION </b>
 
<b> PURIFICATION </b>
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The culture was grown at 16℃ overnight (14-16 hours) and spun down at 4C, 5000 rpm, for 15 mins. The pellet was resuspended using Lysis buffer and were sonicated (at 60% amplitude with a 2 sec ON, 5 sec OFF cycle) until the solution turned slightly brown and translucent. The solution was then spun down (12000 rpm for 25 minutes at 4C), and the supernatant, which contained our protein, was purified using Ni-NTA beads and eluted using increasing concentrations of Imidazole.
 
The culture was grown at 16℃ overnight (14-16 hours) and spun down at 4C, 5000 rpm, for 15 mins. The pellet was resuspended using Lysis buffer and were sonicated (at 60% amplitude with a 2 sec ON, 5 sec OFF cycle) until the solution turned slightly brown and translucent. The solution was then spun down (12000 rpm for 25 minutes at 4C), and the supernatant, which contained our protein, was purified using Ni-NTA beads and eluted using increasing concentrations of Imidazole.
 
The flow-through, the different elutes, and cracked beads was run on a 12% SDS-PAGE to resolve the different protein bands along with a protein ladder that shows 10 bands with 3 reference bands.
 
The flow-through, the different elutes, and cracked beads was run on a 12% SDS-PAGE to resolve the different protein bands along with a protein ladder that shows 10 bands with 3 reference bands.
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[[image:BBa_K4429008_3.png|500px]]
  
 
<b> WESTERN BLOT </b>
 
<b> WESTERN BLOT </b>
 
To ensure that we loaded equal amounts of protein in all the wells for the Western blot, we performed a Bradford Colorimetry assay.
 
To ensure that we loaded equal amounts of protein in all the wells for the Western blot, we performed a Bradford Colorimetry assay.
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 +
[[image:BBa_K4429008_4.png|500px]]
  
 
Normalised amounts of proteins were loaded and run on an SDS-PAGE. The proteins were expressed under different conditions - scFv expressed at 16C, scFv expressed at 25C, scFv expressed at 30C and scFv-FcRnBp (NeoFv) expressed at 16C.
 
Normalised amounts of proteins were loaded and run on an SDS-PAGE. The proteins were expressed under different conditions - scFv expressed at 16C, scFv expressed at 25C, scFv expressed at 30C and scFv-FcRnBp (NeoFv) expressed at 16C.
 
The proteins were transferred to a PVDF membrane and probed using an anti-His primary antibody. The blot was imaged using chemiluminescence.
 
The proteins were transferred to a PVDF membrane and probed using an anti-His primary antibody. The blot was imaged using chemiluminescence.
  
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[[image:BBa_K4429008_5.png|500px]]
  
 
<b> DESIGN OF EXPERIMENTS </b>
 
<b> DESIGN OF EXPERIMENTS </b>
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This design of experiments was created to characterise the influence of factors in the yield of the scFv, and the interactions of different factors. The gels, the outputs and the analysis is presented below.  
 
This design of experiments was created to characterise the influence of factors in the yield of the scFv, and the interactions of different factors. The gels, the outputs and the analysis is presented below.  
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[[image:BBa_K4429008_9.png|500px]]
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[[image:BBa_K4429008_11.png|500px]]
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[[image:BBa_K4429008_8.png|500px]]
  
  
 
The final yield of 379 ug/ml was measured through a BSA standard curve plotted through SDS PAGE
 
The final yield of 379 ug/ml was measured through a BSA standard curve plotted through SDS PAGE
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The interactions observed were not significant
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[[image:BBa_K4429008_6.png|500px]]
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[[image:BBa_K4429008_7.png|500px]]
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[[image:BBa_K4429008_10.png|500px]]
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The interactions observed were not found to be significant
  
 
<!-- Add more about the biology of this part here
 
<!-- Add more about the biology of this part here
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<span class='h3bb'>Sequence and Features</span>
 
<span class='h3bb'>Sequence and Features</span>
 
<partinfo>BBa_K4429008 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K4429008 SequenceAndFeatures</partinfo>
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===References===
 
===References===

Latest revision as of 15:38, 12 October 2022


scFv C10: EDE1

This part codes for the scFv version of the C10 antibody. C10 is an antibody that targets the envelope dimer epitope (EDE) of the Dengue virus[1]. C10 is a highly potent, pan-neutralising antibody against Dengue. An scFv is an antibody fragment that consists of just the variable regions of the antibody connected by a flexible linker.

Another version of an antibody fragment derived from scFvs is the NeoFv, of which BBa_K4429009 is an example.

-

Characterisation

CLONING

The scFv-pUCIDT-KAN and pET-21b vector were both digested with Nde1 and Xho1, run on a 0.8% agarose gel, the DNA fragments extracted and ligated. To check for positive clones, we performed alkaline lysis on the primaries made from the 1:3 plate. A 0.8% agarose gel was run with the suspected positive clones along with the undigested empty vector. The positive clones are expected to show an upward shift of about 800 bp.

Results: The cloning was successful.

BBa K4429008 0.png

BBa K4429008 1.png


PURIFICATION Purification The culture was grown at 16℃ overnight (14-16 hours) and spun down at 4C, 5000 rpm, for 15 mins. The pellet was resuspended using Lysis buffer and were sonicated (at 60% amplitude with a 2 sec ON, 5 sec OFF cycle) until the solution turned slightly brown and translucent. The solution was then spun down (12000 rpm for 25 minutes at 4C), and the supernatant, which contained our protein, was purified using Ni-NTA beads and eluted using increasing concentrations of Imidazole. The flow-through, the different elutes, and cracked beads was run on a 12% SDS-PAGE to resolve the different protein bands along with a protein ladder that shows 10 bands with 3 reference bands.

BBa K4429008 3.png

WESTERN BLOT To ensure that we loaded equal amounts of protein in all the wells for the Western blot, we performed a Bradford Colorimetry assay.

BBa K4429008 4.png

Normalised amounts of proteins were loaded and run on an SDS-PAGE. The proteins were expressed under different conditions - scFv expressed at 16C, scFv expressed at 25C, scFv expressed at 30C and scFv-FcRnBp (NeoFv) expressed at 16C. The proteins were transferred to a PVDF membrane and probed using an anti-His primary antibody. The blot was imaged using chemiluminescence.

BBa K4429008 5.png

DESIGN OF EXPERIMENTS

We ran 15 sets of experiments on three factors:

Temperature post-induction: We varied this between 16C and 37C. From our literature review, the ideal temperature and its interactions was bound to lie between these values. IPTG concentration: Varying from 0.1 to 1mM Optical Density of the culture at induction: Varying from 0.5 to 1

This design of experiments was created to characterise the influence of factors in the yield of the scFv, and the interactions of different factors. The gels, the outputs and the analysis is presented below.


BBa K4429008 9.png

BBa K4429008 11.png


BBa K4429008 8.png


The final yield of 379 ug/ml was measured through a BSA standard curve plotted through SDS PAGE


BBa K4429008 6.png

BBa K4429008 7.png

BBa K4429008 10.png


The interactions observed were not found to be significant

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
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]

References

  • 1] Dejnirattisai, W., Wongwiwat, W., Supasa, S. et al. A new class of highly potent, broadly neutralizing antibodies isolated from viremic patients infected with dengue virus. Nat Immunol 16, 170–177 (2015). https://doi.org/10.1038/ni.3058