Difference between revisions of "Part:BBa K2876014"

Revision as of 02:37, 18 October 2018

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).

Figure 1: Single Chain Antibody Two Hybrid Protein Detection System

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.

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.

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