Difference between revisions of "Part:BBa K4429002"
 
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Generates a full-length chimeric IgG-IgY antibody against maltose-binding protein in when cloned into a pET-21b vector. Check design page for more specific information.  
 
Generates a full-length chimeric IgG-IgY antibody against maltose-binding protein in when cloned into a pET-21b vector. Check design page for more specific information.  
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===Characterisation===
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<b> CLONING </b>
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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 2200 bp.
 +
As a further test for confirmation, a restriction digest with HindIII was run. HindIII is a single cutter for the positive clone, and a non-cutter for the empty vector. A 0.8% agarose gel was run with the products of the restriction digest. A positive clone would be linearized by HindIII.
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We were successful in cloning the gene for the IgY-IgG into our vector. We have confirmed the same by having the plasmid sequenced.
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[[image:BBa_K4429015_0.png|500px]]
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[[image:BBa_K4429015_1.png|500px]]
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<b> Expression check</b>
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We observed bands of varying intensities corresponding to the different concentrations of IPTG and determined that a IPTG concentration of 0.5mM is ideal for maximum yield of protein.
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[[image:BBa_K4429015_2.png|500px]]
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<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.
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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_K4429015_3.png|500px]]
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For the anti-MBP IgY antibody, we got fragmented bands when we ran a SDS-PAGE. This was possibly be due to working with an improper protease inhibitor.
  
 
<!-- Add more about the biology of this part here
 
<!-- Add more about the biology of this part here

Latest revision as of 03:47, 14 October 2022


Full Length Anti-MBP Recombinant IgY

Generates a full-length chimeric IgG-IgY antibody against maltose-binding protein in when cloned into a pET-21b vector. Check design page for more specific information.

Characterisation

CLONING

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 2200 bp. As a further test for confirmation, a restriction digest with HindIII was run. HindIII is a single cutter for the positive clone, and a non-cutter for the empty vector. A 0.8% agarose gel was run with the products of the restriction digest. A positive clone would be linearized by HindIII. We were successful in cloning the gene for the IgY-IgG into our vector. We have confirmed the same by having the plasmid sequenced.


BBa K4429015 0.png

BBa K4429015 1.png

Expression check

We observed bands of varying intensities corresponding to the different concentrations of IPTG and determined that a IPTG concentration of 0.5mM is ideal for maximum yield of protein.

BBa K4429015 2.png

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 K4429015 3.png

For the anti-MBP IgY antibody, we got fragmented bands when we ran a SDS-PAGE. This was possibly be due to working with an improper protease inhibitor.

Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal XbaI site found at 739
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal XbaI site found at 739
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal XbaI site found at 739
  • 1000
    COMPATIBLE WITH RFC[1000]