Difference between revisions of "Part:BBa K554005"
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==Our IL-12 sequence== | ==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. | + | :*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. |
+ | |||
+ | You can find below a tridimensional structure of human Interleukin 12, with both p35 (chain A) and p40 (chain B) chains. This is a jmol applet, in which you can interactively see the protein format: | ||
+ | <html> | ||
+ | <script src="http://www.cbi.cnptia.embrapa.br/jmol/Jmol.js" type="text/javascript"></script> | ||
+ | <script type="text/javascript"> | ||
+ | jmolInitialize("http://www.cbi.cnptia.embrapa.br/jmol/"); | ||
+ | jmolApplet(400, "load http://www.cbi.cnptia.embrapa.br/jmol/files/3OZZ.pdb.gz;"); | ||
+ | jmolBr(); | ||
+ | |||
+ | jmolButton("restrict not dna; restrict not protein; restrict not HOH; restrict not ligand; dots off; selectionhalos off; center selected; zoom 100; select all; wireframe; select all; color chain; select Cu or Cu1 or Zn or fe or Ca or Cl or Mn or Mg or na or Br or SO4 or ligand; color magenta selected; cpk; select all", "Stick view", "Stick view") | ||
+ | |||
+ | jmolButton("restrict not HOH; select all; wireframe off;dots off; ribbon; color structure; zoom 100; center selected; label off; backbone off; selectionhalos off; color structure; select Cu or Cu1 or Zn or fe or Ca or Cl or Mn or Mg or na or Br or SO4 or ligand; cpk; select ligand; wireframe 75; select all; Hbonds off; SSbonds off; select ligand; ribbon off", "Ribbon view", "Ribbon view") | ||
+ | |||
+ | jmolButton("restrict not dna; restrict not protein; restrict not HOH; restrict not ligand; dots off; selectionhalos off; center selected; zoom 100; select all; wireframe; select all; color chain; backbone 50; wireframe off; select Cu or Cu1 or Zn or fe or Ca or Cl or Mn or Mg or na or Br or SO4 or ligand; cpk; select ligand; wireframe 75; select all; Hbonds off; SSbonds off; select ligand; backbone off", "Wireframe view", "Wireframe view") | ||
+ | |||
+ | jmolButton("spin y" ,"spin y"); | ||
+ | jmolButton("spin off" ,"spin off"); | ||
+ | </script> | ||
+ | |||
+ | <br /> | ||
+ | </html> | ||
==A synthesis trigger: promoter flhDC== | ==A synthesis trigger: promoter flhDC== |
Revision as of 20:43, 26 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
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 1409
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
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.
You can find below a tridimensional structure of human Interleukin 12, with both p35 (chain A) and p40 (chain B) chains. This is a jmol applet, in which you can interactively see the protein format:
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.
Schema:
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: