Difference between revisions of "Part:BBa K2876014"
(→‎MIT MAHE 2020)
 
(14 intermediate revisions by 2 users not shown)
Line 3: Line 3:
 
<partinfo>BBa_K2876014 short</partinfo>
 
<partinfo>BBa_K2876014 short</partinfo>
  
−
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.  
+
This sequence produces protein interleukin-1B, a human protein produced in response to illness and fever. We selected IL1B because it is an important human immunoprotein with multiple well-characterized antibodies. We fused two single chain antibodies targeting different IL1B epitopes (BBa_K2876015) (https://parts.igem.org/Part:BBa_K2876015) and  BBa_K2876011 (https://parts.igem.org/Part:BBa_K2876015) to the lambda repressor and alpha subunit of RNA-polymerase III (BBa_K2876002) (https://parts.igem.org/Part:BBa_K2876002) described in Dove, Joung, and Hochschild in order to create two fusion proteins capable of activating transcription in the presence of IL1B (Figure 1). For our reporter we used the previously described pOL2-62 from Dove, Joung, and Hochschild fused to a RFP (BBa_K2876000: (https://parts.igem.org/Part:BBa_K2876000).  
 +
 
 +
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 1).
 +
 
 +
[[File:SCAP2h_protein.png|400px]]
 +
 
 +
 
 +
Figure 1: Single Chain Antibody Two Hybrid Protein Detection System
 +
 
 +
 
 +
 
 +
[[File:IL1B graph.png|800px]]
 +
 
 +
Figure 2: 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.
 +
 
 +
 
 +
[[File:IL1B ELISA Pre-stop.jpg|400px]]
 +
 
 +
Figure 3: 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.
 +
 
 +
 
 +
[[File:IL1B ELISApoststop.png|400px]]
 +
 
 +
Figure 4: 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.
  
−
[[File:IL1B ELISA Pre-stop.jpg]]
 
  
−
Figure 1: 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 to most 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.
 
  
−
[[File:IL1B ELISApoststop.png]]
 
−
Figure 2: 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 to most 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.
 
  
 
<!-- Add more about the biology of this part here
 
<!-- Add more about the biology of this part here
Line 24: Line 43:
 
<partinfo>BBa_K2876014 parameters</partinfo>
 
<partinfo>BBa_K2876014 parameters</partinfo>
 
<!-- -->
 
<!-- -->
 +
 +
==MIT_MAHE 2020==
 +
'''Summary'''
 +
 +
Interleukin-1β (IL-1β), belongs to the interleukin-1 (IL-1) cytokine family. It is a pro-inflammatory cytokine and key in mediating body's responses to microbial infection, immunological reactions, and tissue injury. Processing of IL-1β precursor to active form can be done using various proteases. It is involved in mediating inflammation, initiating or increasing a wide variety of non-structural, function associated genes that are usually expressed during inflammation. There are now full cDNA sequences and genomic organisation of IL-1 beta taken from bird, amphibian, bony fish and cartilaginous fish, with many of these genes having been obtained using an homology cloning approach.
 +
 +
==References==
 +
 +
1. Husain, M., Bird, S., van Zwieten, R., Secombes, C. J., & Wang, T. (2012). Cloning of the IL-1β3 gene and IL-1β4 pseudogene in salmonids uncovers a second type of IL-1β gene in teleost fish. Developmental and comparative immunology, 38(3), 431–446. https://doi.org/10.1016/j.dci.2012.07.010
 +
                                                                                                                                                             
 +
2. Bird, S., Zou, J., Wang, T., Munday, B., Cunningham, C., & Secombes, C. J. (2002). Evolution of interleukin-1beta. Cytokine & growth factor reviews, 13(6), 483–502. https://doi.org/10.1016/s1359-6101(02)00028-x

Latest revision as of 17:53, 23 October 2020


IL1B

This sequence produces protein interleukin-1B, a human protein produced in response to illness and fever. We selected IL1B because it is an important human immunoprotein with multiple well-characterized antibodies. We fused two single chain antibodies targeting different IL1B epitopes (BBa_K2876015) (https://parts.igem.org/Part:BBa_K2876015) and BBa_K2876011 (https://parts.igem.org/Part:BBa_K2876015) to the lambda repressor and alpha subunit of RNA-polymerase III (BBa_K2876002) (https://parts.igem.org/Part:BBa_K2876002) described in Dove, Joung, and Hochschild in order to create two fusion proteins capable of activating transcription in the presence of IL1B (Figure 1). For our reporter we used the previously described pOL2-62 from Dove, Joung, and Hochschild fused to a RFP (BBa_K2876000: (https://parts.igem.org/Part:BBa_K2876000).

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 1).

SCAP2h protein.png


Figure 1: Single Chain Antibody Two Hybrid Protein Detection System


IL1B graph.png

Figure 2: 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 3: 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 4: 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.



Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 603
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


MIT_MAHE 2020

Summary

Interleukin-1β (IL-1β), belongs to the interleukin-1 (IL-1) cytokine family. It is a pro-inflammatory cytokine and key in mediating body's responses to microbial infection, immunological reactions, and tissue injury. Processing of IL-1β precursor to active form can be done using various proteases. It is involved in mediating inflammation, initiating or increasing a wide variety of non-structural, function associated genes that are usually expressed during inflammation. There are now full cDNA sequences and genomic organisation of IL-1 beta taken from bird, amphibian, bony fish and cartilaginous fish, with many of these genes having been obtained using an homology cloning approach.

References

1. Husain, M., Bird, S., van Zwieten, R., Secombes, C. J., & Wang, T. (2012). Cloning of the IL-1β3 gene and IL-1β4 pseudogene in salmonids uncovers a second type of IL-1β gene in teleost fish. Developmental and comparative immunology, 38(3), 431–446. https://doi.org/10.1016/j.dci.2012.07.010

2. Bird, S., Zou, J., Wang, T., Munday, B., Cunningham, C., & Secombes, C. J. (2002). Evolution of interleukin-1beta. Cytokine & growth factor reviews, 13(6), 483–502. https://doi.org/10.1016/s1359-6101(02)00028-x