Difference between revisions of "Part:BBa K3110051"
 
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<partinfo>BBa_K3110051 short</partinfo>
 
<partinfo>BBa_K3110051 short</partinfo>
  
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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.
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This part contains the lldRO1-J23117-lldRO2 attached to a strong RBS, YebF-IL12-FLAG and a double terminator.  
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===Usage and Biology===
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<partinfo>BBa_K3110051 parameters</partinfo>
 
<partinfo>BBa_K3110051 parameters</partinfo>
 
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<h2>Usage and Biology</h2>
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The regulatory element lldR binds to the lldRO1 and lldRO2 (the operator regions) and inhibits 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 the lldR complex with L-Lactate remains bound to LldRO1, acting as a transcriptional activator. IL12 is an anti-inflammatory cytokine involved in the regulation of cell-mediated immune responses. Through a complex signaling cascade, IL12 activates our immune cells to reduce tumor growth. Its production is under the control of the 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 the 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 Flag antibodies.
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<h1>Usage and Biology</h1>
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The regulatory element lldR binds to the lldRO1 and lldRO2 (the operator regions) and inhibits 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 to the promoter. Upon binding of L-lactate to lldR, this transcriptional suppression is lost and the lldR complex with L-lactate remains bound to LldRO1, acting as a transcriptional activator.<br>IL12 is an anti-inflammatory cytokine involved in the regulation of cell-mediated immune responses. Through a complex signalling cascade, IL12 activates our immune cells to reduce tumor growth. Its production is under the control of the lactate operon's 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 <i>E. coli</i>. We have attached the IL12 sequence in the C-terminus of YebF so that IL12 also gets secreted out of <i>E. coli</i> along with YebF. A FLAG tag is attached downstream to YebF so that its secretion outside the cells can be detected using FLAG antibodies.
  
 
<h1>Characterization of the construct</h1>
 
<h1>Characterization of the construct</h1>
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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, IL12 was placed under the control of O1PO2. This also gave specificity to the production of IL12 only at desired concentrations of lactate. A secretory protein YebF was cojugated to IL12 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 IL12 outside the cells.
 
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<h3>Characterization</h3>
 
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<ul>
 
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<li>We chose BL21(DE3) as our chassis and transformed the plasmid containing our construct in BL21(DE3). We cultured them in LB (with Chloramphenicol of concentration 34 ng/ul) for 12 hours at 37dC, 200 r.p.m. in two separate conical flasks.</li>
 
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<li>One of them was induced with lactate at an OD of 0.4 while the other was left uninduced.</li>
 
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<li>After 12 hours, the culture medium and the cell pellet were then taken for western blot after undergoing sample preparation.</li>
 
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<li>The samples were then run on a 15% polyacrylamide gel and were then transferred onto PVDF membrane. The membrane was then processed with the help of Flag primary antibodies, anti mouse conjugated with HRP secondary antibodies and Blocking solution.</li>
 
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<li>For imaging, luminol based solution was used, as our secondary antibody was conjugated with HRP. </li></ul>
 
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<h5>SOEing of O1pO2-Strong RBS- YebF IL12 FLAG-Terminator</h5>
 
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<ul>
 
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This construct was a part of our IL12 secretory system. While positive clones were obtained, IL12 was not detected either in the cell pellet or in the supernatant. We hypothesise a faulty SOEing that could have caused errors in our promoter or RBS regions.
 
  
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The following gel image shows the band obtained after SOEing of O1pO2-Strong RBS- YebF IL12 FLAG-Terminator. As expected, a band of 2310 bp was obtained.
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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, IL12 was placed under the control of O1PO2. This should give specificity to the production of IL12 only at desired concentrations of lactate. A secretory protein YebF was conjugated to IL12 so as to facilitate its secretion outside the cells once it is produced in the cancer microenvironment. The FLAG tag was attached downstream to detect the secretion of IL12 outside the cells.
  
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The obtained construct was ligated to pSB1C3 plasmid and transformed in DH5alpha.
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<h3>Experimental Procedure</h3>
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<h6>Selection of positive colonies</h6>
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Colony PCR was done to select the positive colonies. The following gel image shows the band obtained after performing colony PCR with VF2 and VR primers. 5 out of 9 colonies gave a band at 2.5 kb as expected.
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<ul>
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<h7> Confirmation of the presence of correct plasmids in the selected colonies </h7>
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<li>We chose BL21(DE3) as our chassis and transformed the plasmid containing our construct in BL21(DE3). We cultured them in LB (with Chloramphenicol at a concentration of 34 ng/ul) for 12 hours at 37°C, 200 r.p.m. in two separate conical flasks.</li>
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The following gel image shows the bands obtained on linearisation and insert release. As expected, a band of 4.4 kb was obtained on linearisation and two bands of 2.3 kb and 2.1 kb were obtained on insert release.
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<li>One of them was induced with L-lactate at an OD of 0.4 while the other was left uninduced.</li>
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<li>After 12 hours, the culture medium and the cell pellet were then taken for western blot after undergoing sample preparation which included the concentration of supernatant using a 10 kDa MWCO filter.</li>
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<li>The samples were then run on a 15% polyacrylamide gel and followed by transferring onto a PVDF membrane with a pore size of 0.2 µm. The membrane was then probed with the help of FLAG primary antibodies (raised in mouse) and a secondary anti-mouse antibody conjugated with HRP.</li>
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<li>For imaging, luminol-peroxide based solution was used, as our secondary antibody was conjugated with HRP. </li>
 
</ul>
 
</ul>
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<h3>Results</h3>
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[[File:T--IISER Tirupati--IL12_1.jpeg|thumb|center|700px|Figure 1. SDS PAGE of ConsP-Strong RBS-YebF-IL12-FLAG (Both Supernatant and Pellet)]]
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In the case of constitutive promoter upstream to YebF-IL12, we can see a band at around 74 KDa (expected size) in the pellet fraction. The band is present both in the control and test samples. However, no band was observed in the supernatant.<br><br>
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[[File:T--IISER Tiruapti--IL12_2.jpeg|thumb|center|700px|Figure 1. SDS PAGE of O1PO2-Strong RBS-YebF-IL12-FLAG (Both Supernatant and Pellet)]]
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In the case of O1PO2 upstream to YebF-IL12, we can see a band at around 74 KDa in the supernatant and cell pellet both with and without lactate induction. <br>
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The SDS-PAGES were not very conclusive and we  decided to proceed with Western Blot analysis to confirm if the band corresponded to YEBF-IL12.<br><br>
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[[File:T--IISER Tirupati--IL12_3.jpeg|thumb|center|700px|Figure 1. Western BLot of O1PO2-Strong RBS-YebF-IL12-FLAG (Both Supernatant and Pellet) using FLAG-Antibody (raised in mouse) and Anti-Mouse-Antibody (HRP conjugated)]]
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We didn't observe any band at around 74 KDa. Hence we can conclude that the protein is nor being produced only. We suspected that there might have been SOEing errors in the steps of cloning and this requires further investigation.

Latest revision as of 02:47, 22 October 2019


lldRO1-J23117-lldRO2 Strong RBS YebF-IL12

This part contains the lldRO1-J23117-lldRO2 attached to a strong RBS, YebF-IL12-FLAG and a double terminator.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 78
    Illegal NheI site found at 101
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


Usage and Biology

The regulatory element lldR binds to the lldRO1 and lldRO2 (the operator regions) and inhibits 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 to the promoter. Upon binding of L-lactate to lldR, this transcriptional suppression is lost and the lldR complex with L-lactate remains bound to LldRO1, acting as a transcriptional activator.
IL12 is an anti-inflammatory cytokine involved in the regulation of cell-mediated immune responses. Through a complex signalling cascade, IL12 activates our immune cells to reduce tumor growth. Its production is under the control of the lactate operon's 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 the 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 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, IL12 was placed under the control of O1PO2. This should give specificity to the production of IL12 only at desired concentrations of lactate. A secretory protein YebF was conjugated to IL12 so as to facilitate its secretion outside the cells once it is produced in the cancer microenvironment. The FLAG tag was attached downstream to detect the secretion of IL12 outside the cells.

Experimental Procedure

  • We chose BL21(DE3) as our chassis and transformed the plasmid containing our construct in BL21(DE3). We cultured them in LB (with Chloramphenicol at a concentration of 34 ng/ul) for 12 hours at 37°C, 200 r.p.m. in two separate conical flasks.
  • One of them was induced with L-lactate at an OD of 0.4 while the other was left uninduced.
  • After 12 hours, the culture medium and the cell pellet were then taken for western blot after undergoing sample preparation which included the concentration of supernatant using a 10 kDa MWCO filter.
  • The samples were then run on a 15% polyacrylamide gel and followed by transferring onto a PVDF membrane with a pore size of 0.2 µm. The membrane was then probed with the help of FLAG primary antibodies (raised in mouse) and a secondary anti-mouse antibody conjugated with HRP.
  • For imaging, luminol-peroxide based solution was used, as our secondary antibody was conjugated with HRP.

Results

Figure 1. SDS PAGE of ConsP-Strong RBS-YebF-IL12-FLAG (Both Supernatant and Pellet)

In the case of constitutive promoter upstream to YebF-IL12, we can see a band at around 74 KDa (expected size) in the pellet fraction. The band is present both in the control and test samples. However, no band was observed in the supernatant.

Figure 1. SDS PAGE of O1PO2-Strong RBS-YebF-IL12-FLAG (Both Supernatant and Pellet)

In the case of O1PO2 upstream to YebF-IL12, we can see a band at around 74 KDa in the supernatant and cell pellet both with and without lactate induction.
The SDS-PAGES were not very conclusive and we decided to proceed with Western Blot analysis to confirm if the band corresponded to YEBF-IL12.

Figure 1. Western BLot of O1PO2-Strong RBS-YebF-IL12-FLAG (Both Supernatant and Pellet) using FLAG-Antibody (raised in mouse) and Anti-Mouse-Antibody (HRP conjugated)

We didn't observe any band at around 74 KDa. Hence we can conclude that the protein is nor being produced only. We suspected that there might have been SOEing errors in the steps of cloning and this requires further investigation.