Difference between revisions of "Part:BBa K3110051"
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<h1>Characterization of the construct</h1> | <h1>Characterization of the construct</h1> | ||
The lactate sensitive promoter lldPRDp was characterised and sfgfp was observed to give better fluorescence intensity under O1PO2 than when it was placed under the control of a constitutive promoter. So, IL-12 was placed under the control of O1PO2. This also gave specificity to the production of IL-12 only at desired concentrations of lactate. A secretory protein YebF was cojugated to IL-12 so as to promote its secretion outside the cells once it is produced in the cancer microenvironment. The flag tag was attached downstream to detect the secretion of IL-12 outside the cells. | The lactate sensitive promoter lldPRDp was characterised and sfgfp was observed to give better fluorescence intensity under O1PO2 than when it was placed under the control of a constitutive promoter. So, IL-12 was placed under the control of O1PO2. This also gave specificity to the production of IL-12 only at desired concentrations of lactate. A secretory protein YebF was cojugated to IL-12 so as to promote its secretion outside the cells once it is produced in the cancer microenvironment. The flag tag was attached downstream to detect the secretion of IL-12 outside the cells. | ||
| + | <h3>Characterization</h3> | ||
| + | <ul> | ||
| + | <li>We chose BL21(DE3) as our chassis and transformed the plasmid containing our construct in BL21(DE3). We cultured them in M9-Glycerol(with Chloramphenicol of concentration 34 ng/ul) for 12 hours at 37dC, 200 r.p.m. in two separate conical flasks. One was induced with lactate at an OD of 0.4 while the other was left uninduced. The culture medium and the cell pellet were then taken for western blot after undergoing sample preparation. The samples were then run on a 15% gel and were then transferred onto PVDF membrane. The membrane was then processed with the help of anti-Flag primary antibodies, anti mouse conjugated with HRP secondary antibodies and Blocking solution. For imaging, luminol based solution was used as our secondary antibody was conjugated with HRP. </li> | ||
Revision as of 15:43, 20 October 2019
lldRO1-J23117-lldRO2 Strong RBS YebF-IL12
lldRO1 and lldRO2 are the operator regions which bind to the regulator element lldR and inhibit transcription. Upon binding of L-Lactate to lldR, however, this transcriptional suppression is lost and instead lldR complex with L-Lactate remains bound to lldRO1 acting as a transcriptional activator. By using RBS of different strengths we are trying to find out the optimum construct which will give output (in this case the output is YebF-IL12) that can efficiently discriminate cancer vs non-cancer micro-environments and will lead to secretion of IL12 (natural immuno-modulator) and eventually reduction of tumour size.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 78
Illegal NheI site found at 101 - 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
Usage and Biology
The regulatory element lldR binds to the lldRO1 and lldRO2 (the operator regions) and inhibit transcription. J23117 is a promoter intercalated between the operators. lldR dimer represses the transcription possibly by forming a DNA loop which doesn’t allow the RNA polymerase to bind the promoter. Upon binding of L-Lactate to LldR, however, this transcriptional suppression is lost and instead lldR complex with L-Lactate remains bound to LldRO1 acting as a transcriptional activator. Interleukin-12. IL-12 is an anti-inflammatory cytokine, involved in the regulation of cell-mediated immune responses. Through a set of complex signaling cascade IL-12 activates our immune cells to reduce tumor growth. Its production is under the control of lactic acid promoter, lldPRDp which has been optimised to respond specifically to lactic acid concentrations in the milieu of colon cancer cells. YebF is a secretory protein present in E.coli. We have attached IL12 sequence in the C-terminus of YebF so that IL12 also gets secreted out of E.coli along with YebF. A flag tag is attached downstream to YebF so that its secretion outside the cells can be detected using anti-Flag antibodies.
Characterization of the construct
The lactate sensitive promoter lldPRDp was characterised and sfgfp was observed to give better fluorescence intensity under O1PO2 than when it was placed under the control of a constitutive promoter. So, IL-12 was placed under the control of O1PO2. This also gave specificity to the production of IL-12 only at desired concentrations of lactate. A secretory protein YebF was cojugated to IL-12 so as to promote its secretion outside the cells once it is produced in the cancer microenvironment. The flag tag was attached downstream to detect the secretion of IL-12 outside the cells.
Characterization
- We chose BL21(DE3) as our chassis and transformed the plasmid containing our construct in BL21(DE3). We cultured them in M9-Glycerol(with Chloramphenicol of concentration 34 ng/ul) for 12 hours at 37dC, 200 r.p.m. in two separate conical flasks. One was induced with lactate at an OD of 0.4 while the other was left uninduced. The culture medium and the cell pellet were then taken for western blot after undergoing sample preparation. The samples were then run on a 15% gel and were then transferred onto PVDF membrane. The membrane was then processed with the help of anti-Flag primary antibodies, anti mouse conjugated with HRP secondary antibodies and Blocking solution. For imaging, luminol based solution was used as our secondary antibody was conjugated with HRP.