Difference between revisions of "Part:BBa K2876014:Experience"

 
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This experience page is provided so that any user may enter their experience using this part.<BR>Please enter
 
This experience page is provided so that any user may enter their experience using this part.<BR>Please enter
 
how you used this part and how it worked out.
 
how you used this part and how it worked out.
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This sequence produces protein interleukin-1B, a human protein produced during fevers. For our project we detected IL1B with two single-chain antibodies fused to transcription initiating proteins (BBa_K2876001, BBa_K2876002). We validated our IL1B with an ELISA (https://www.thermofisher.com/elisa/product/IL-1-beta-Human-ELISA-Kit/BMS224-2) which proved production of IL1B, and that the IL1B produced was folded properly so that antibodies could bind. Validation of antibody binding was essential to ensure that IL1B could function as the target protein in our Single-Chain Antibody Prokaryotic Two Hybrid Detection System (Figure 4).
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[[File:IL1B graph.png|800px]]
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Figure 1: A graph of the absorbance at 540 nm from an ELISA of DH5-alpha cells transformed to produce IL1B (in orange) compared to the IL1B-Standard (in blue) and the negative control of untransformed DH5-alpha cells (in grey). Protein was extracted using a B-PER protocol. We used the negative control to calculate a null hypothesis for the log of absorbance when there is no protein. H0 Absorbance = Log(0.082) = -1.086. We then did a right-tailed hypothesis test to see if our values of Human Interleukin-1 Beta were statistically significant. The z-score was 21.57; the cutoff for statistical significance was a z-score of 1.895. Therefore, our data was statistically significant and we can accept the alternative hypothesis, Ha: Absorbance =/= -1.806. This leads us to conclude that IL1B protein recognizable by IL1B antibodies is being made.
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[[File:IL1B ELISA Pre-stop.jpg|400px]]
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Figure 2: A photo of the IL1B ELISA showing color change as a function of IL1B presence. Columns 1,2, and 6 show the ELISA standards, from least (row A) to most (row H) diluted. Columns 3, 4, and 5 rows A-D are DH5-alpha cells transformed with our IL1B plasmid. Columns 3, 4, and 5 row E are untransformed DH5-alpha cells.
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[[File:IL1B ELISApoststop.png|400px]]
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Figure 3: A photo of the IL1B ELISA  showing color change as a function of IL1B presence, after stop-solution added. Columns 1,2, and 6 show the ELISA standards, from least (row A) to most (row H) diluted. Columns 3, 4, and 5 rows A-D are DH5-alpha cells transformed with our IL1B plasmid. Columns 3, 4, and 5 row E are untransformed DH5-alpha cells.
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[[File:SCAP2h_protein.png|400px]]
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Figure 4: Single Chain Antibody Two Hybrid Protein Detection System
  
 
===Applications of BBa_K2876014===
 
===Applications of BBa_K2876014===

Revision as of 01:16, 18 October 2018


This experience page is provided so that any user may enter their experience using this part.
Please enter how you used this part and how it worked out.

This sequence produces protein interleukin-1B, a human protein produced during fevers. For our project we detected IL1B with two single-chain antibodies fused to transcription initiating proteins (BBa_K2876001, BBa_K2876002). We validated our IL1B with an ELISA (https://www.thermofisher.com/elisa/product/IL-1-beta-Human-ELISA-Kit/BMS224-2) which proved production of IL1B, and that the IL1B produced was folded properly so that antibodies could bind. Validation of antibody binding was essential to ensure that IL1B could function as the target protein in our Single-Chain Antibody Prokaryotic Two Hybrid Detection System (Figure 4).

IL1B graph.png

Figure 1: A graph of the absorbance at 540 nm from an ELISA of DH5-alpha cells transformed to produce IL1B (in orange) compared to the IL1B-Standard (in blue) and the negative control of untransformed DH5-alpha cells (in grey). Protein was extracted using a B-PER protocol. We used the negative control to calculate a null hypothesis for the log of absorbance when there is no protein. H0 Absorbance = Log(0.082) = -1.086. We then did a right-tailed hypothesis test to see if our values of Human Interleukin-1 Beta were statistically significant. The z-score was 21.57; the cutoff for statistical significance was a z-score of 1.895. Therefore, our data was statistically significant and we can accept the alternative hypothesis, Ha: Absorbance =/= -1.806. This leads us to conclude that IL1B protein recognizable by IL1B antibodies is being made.

IL1B ELISA Pre-stop.jpg


Figure 2: A photo of the IL1B ELISA showing color change as a function of IL1B presence. Columns 1,2, and 6 show the ELISA standards, from least (row A) to most (row H) diluted. Columns 3, 4, and 5 rows A-D are DH5-alpha cells transformed with our IL1B plasmid. Columns 3, 4, and 5 row E are untransformed DH5-alpha cells.

IL1B ELISApoststop.png


Figure 3: A photo of the IL1B ELISA showing color change as a function of IL1B presence, after stop-solution added. Columns 1,2, and 6 show the ELISA standards, from least (row A) to most (row H) diluted. Columns 3, 4, and 5 rows A-D are DH5-alpha cells transformed with our IL1B plasmid. Columns 3, 4, and 5 row E are untransformed DH5-alpha cells.


SCAP2h protein.png


Figure 4: Single Chain Antibody Two Hybrid Protein Detection System

Applications of BBa_K2876014

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