Part:BBa_K3110051

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 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.

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 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.

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 of concentration 34 ng/ul) for 12 hours at 37dC, 200 r.p.m. in two separate conical flasks.
• One of them was induced with 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.
• 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.
• For imaging, luminol 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 case of constitutive promoter upstream to YebF-IL12, we can see a band at around 75 KDa in the supernatant.

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

In case of O1PO2-YebF-IL12 upstream to YebF-IL12, we can see a band at around 75 KDa in the supernatant and cell pellet both with and without lactate induction. To confirm the band corresponded to YEBF-IL12 we performed western blot with Flag antibodies.

Figure 1. Western BLot of O1PO2-Strong RBS-YebF-IL12-FLAG (Both Supernatant and Pellet) using FLAG-Antibody and Anti-FLAG-Antibody

We didn't observe any band at around 75 KDa. So, we can not conclude anything about this part. It is possible that the band at 75 KD in SDS PAGE is because of noise. Due to shortage of time we couldn't resolve the problem.