Difference between revisions of "Part:BBa K554005"

Revision as of 05:38, 22 September 2011

IL12

IL12 is an heterodimeric cytokine, composed of a disulfide-bound 35 kDa subunit (p35) and a 40 kDa subunit (p40) which enhances both innate and adaptative immune system. It is primarily produced by antigen-presenting cells such as dentritic cells.MORALES et al. (2004) established that sustained IL-12 signaling is required for Th1 development: It drives naïve Th0 differentiation towards the Th1 phenotype, which makes it an appropriate answer to a Th2 imbalance. We thus decided to create a completely new biobrick, responsible for Il-12 synthesis.

Sequence and Features

Usage and Biology

A fusion IL-12 protein

• As MORRIS et al. (2008) [4] recollect, “Early approaches in generating recombinant IL-12 involved co-transfection of cells with two different vectors, each expressing one of the two genes (p40 or p35), or expression of the individual genes under the control of separate promoters in a single plasmid or viral vector. Both approaches were disadvantaged due to inefficient expression of the functional heterodimer with the likelihood of p40 homodimer production” (And thus competitive IL-12 antagonist action.)
• In order to counter those difficulties, several research teams reported the expression of a bioactive single chain porcine IL-12 fusion protein, such as LIESCHKE et al. (1997), whose protein comprised the p40 subunit linked by a (Gly, Ser) linker to the p35 subunit from which the first 22 amino acids were deleted. It proved bioactive with an apparent specific activity comparable to recombinant human IL-12.

Our IL-12 sequence

• As successful work (correct synthesis and protein proved bioactive) has already been done on it , we chose to use a fusion construct of human IL-12, similarly as the one used by LIESCHKE et al. (1997) porcine fusion IL-12 protein for our project.

A synthesis trigger: promoter flhDC

• We need a device that only works when needed: recombinant IL-12 expression must not be continuous, it has to be a reaction to a Th2 imbalance and stop when the balance is reestablished. Therefore, we need a promoter that would be a sensible “trigger”.
• As we explained in the sensor part (the bacterial adrenergic sensor), our device will detect a Th2 imbalance via QseB/QseC sensor, that will be activated in response to adrenaline detection. It will then be able to activate the flhDC promoter (SPERANDIO et al. , 2002) that we will be used to control the IL-12 gene expression.

IL-12 secretion

• With this right promoter, a ribosome binding site (RBS), the IL-12 gene, and a terminator, E.Coli is now able to synthesize IL-12 in response to a Th2 imbalance. But, being a bacteria, it doesn’t have the required material to secrete it in the body. Hence we also need to construct a secretion system, that will be described in the dedicated part of this wiki.
• In our “IL-12 synthesis part”, we will just add a secretion signal sequence (HlyA) for IL-12 to be correctly dealt with by the secretion system.

This sequence is used by [http://2011.igem.org/Team:UNICAMP-EMSE_Brazil UNICAMP-EMSE Brazil team] in the [http://2011.igem.org/Team:UNICAMP-EMSE_Brazil/Project/Device1 Adrenaline sensor device] ("Device 1", which senses Cathecolamine and AI-3 levels and responds by producing and secreting IL-11. This part is shown in red in the following schema:

References

1. Hidalgo E, Leautaud V, Demple B (1998) The redox-regulated SoxR protein acts from a single DNA site as a repressor and an allosteric activator. The EMBO Journal 17(9)2629–2636 [http://www.ncbi.nlm.nih.gov/pubmed/?term=9564045%20 Link to Pubmed]