Team:IISER Tirupati/Results
Construct 1: O1PO2-Strong RBS with sfGFP-terminator (BBa_K3110040)
SOEing of O1PO2-Strong RBS with sfGFP-terminator
The following gel image shows the band obtained after SOEing O1PO2-Strong RBS with sfGFP-terminator. As expected, a band of 1061 bp was obtained.
The obtained construct was then ligated into pSB1C3 plasmid and transformed into DH5alpha.
Selection of positive colonies
Colony PCR was done to select the positive colonies.
The following gel image shows the band obtained after performing colony PCR with Prefix and VR primers. 1 colony out of 4 gave band at 1.2 kb as expected.
Confirmation of the presence of correct plasmids in the selected colonies
Plasmids from the positive colonies were isolated. Linearisation of the plasmid and insert release using the appropriate restriction enzyme were done to confirm that the selected colonies are positive.
The following gel image shows the bands obtained on linearisation and insert release. As expected, a band of 3.1 kb was obtained on linearisation and 2 bands of 1061 bp and 2.1 kb was obtained upon insert release.
Construct 2: Constitutive P-Strong RBS-sfGFP-terminator (BBa_K3110043)
SOEing of Constitutive P-Strong RBS with sfGFP-terminator
The obtained construct was ligated into pSB1C3 plasmid and transformed in DH5alpha.
Selection of positive colonies
Positive colonies were selected by performing colony PCR using prefix and VR primers. 4 colonies out of 5 gave band at 1.1 kb as expected.
Confirmation of the presence of correct plasmids in the selected colonies
As expected, a band of 3 kb was obtained on linearisation and 2 bands of 950 bp and 2.1 kb was obtained on insert release.
Construct 3: O1PO2-Weak RBS-sfGFP-Terminator (BBa_K3110042)
SOEing of O1PO2-Weak RBS with sfGFP-Terminator
This construct was designed to give us a weaker expression of sfGFP as compared to the previous constructs. O1PO2-Weak RBS was SOEed with sfGFP-Terminator and as expected a band of 1060 bp was obtained.
The obtained construct was ligated into pSB1C3 plasmid and transformed in DH5alpha.
Selection of positive colonies
Colony PCR was done to select the positive colonies
The following gel image shows the band obtained after performing colony PCR with prefix and VR primers. 10 colonies out of 10 gave band at 1.2 kb as expected.
Confirmation of the presence of correct plasmids in the selected colonies
As expected, a band of 3.1 kb was obtained on linearisation and 2 band of 1060 bp and 2.1 kb was obtained on insert release.
Construct 4: Constitutive P-Weak RBS-sfGFP-Terminator (BBa_K3110045)
SOEing of Constitutive P-Weak RBS with sfGFP-Terminator
This construct was designed as a control for construct 3.The following gel image shows the band obtained after SOEing Constitutive P-Strong RBS with sfGFP-Terminator. As expected, a band of 950 bp was obtained.
The obtained construct was ligated into pSB1C3 plasmid and transformed in DH5alpha
Selection of positive colonies
Colony PCR was done to select the positive colonies
The following gel image shows the band obtained after performing colony PCR with prefix and VR primers. 9 colonies out of 9 gave band at 1.1 kb as expected.
Confirmation of the presence of correct plasmids in the selected colonies
Plasmids from the positive colonies were isolated. As expected, a band of 3 kb was obtained on linearisation and 2 band of 950 bp and 2.1 kb was obtained on insert release.
Construct 5: O1PO2-Strong RBS -YebF IL12 FLAG-Terminator (BBa_K3110051)
SOEing of O1PO2-Strong RBS -YebF IL12 FLAG-Terminator
This construct was a part of our IL12 secretory system. While positive clones were obtained, IL12 wasn’t detected either in the cell pellet or in the supernatant. We hypothesise a faulty SOEing that could’ve caused a errors in our promoter or RBS regions.
The following gel image shows the band obtained after SOEing O1PO2-Strong RBS with -YebF IL12 FLAG-Terminator. As expected, a band of 2310 bp was obtained.
The obtained construct was ligated to pSB1C3 plasmid and transformed in DH5alpha.
Selection of positive colonies
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 band at 2.5 kb as expected.
Confirmation of the presence of correct plasmids in the selected colonies
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 2 band of 2.3 kb and 2.1 kb were obtained on insert release.
Construct 6: Constitutive P-Strong RBS -YebF IL12 FLAG-Terminator (BBa_K3110054)
SOEing of Const Strong P-Strong RBS -YebF IL12 FLAG-Terminator
This construct was designed as a control for the construct 5, and was supposed to show higher protein expression. The following gel image shows the band obtained after SOEing Const. Strong P-Strong RBS with -YebF IL12-Terminator. As expected, a band of 2205 bp was obtained.
The obtained construct was ligated to psB1C3 plasmid and transformed in DH5alpha.
Selection of positive colonies
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. 4 colonies out of 10 gave band at 2.5 kb as expected.
Confirmation of the presence of correct plasmids in the selected colonies
Linearisation of the plasmid and insert release using the corresponding primers were done to confirm that the selected colonies are positive.
The following gel image shows the bands obtained on linearisation and insert release. As expected, a band of 4.3 kb was obtained on linearisation and 2 band of 2.2 kb and 2.1 kb was obtained on insert release.
Construct 7: Medium P - Strong RBS - lldD -Terminator (BBa_K3110007)
Confirmation of the presence of correct plasmids in the selected colonies
The construct was received from twist bioscience was ligated to pSB3K3 and transformed in DH5alpha.The following gel image shows the bands obtained on linearisation and insert release. As expected, a band of 4.2 kb was obtained on linearisation and 2 band of 1500 bp and 2.7 kb was obtained on insert release.
Construct 8: Constitutive P - Strong RBS - FimHRPMrel -Terminator (BBa_K3110047)
SOEing of Constitutive P - Strong RBS - FimHRPMrel -Terminator
This construct was made to help our bacteria secrete our colon cancer homing peptide at the tip of its fimbriae. The constitutive strong promoter used here is J23100.
The following gel image shows the band obtained after SOEing Constitutive P-Strong RBS with FimHRPMrel -Terminator. As expected, a band of 1250 bp was obtained.
The obtained construct was ligated to pSB1C3 plasmid and transformed in DH5alpha.
Selection of positive colonies
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. No colonies gave band at the expected size of 1.4 kb.
Overlaid chromatogram of standard solutions.
This graph tells us about the retention time of L-lactate in the mobile phase which was used. As two peaks were observed, we collected the elute corresponding to the two retention times and performed a mass spectrometry (data not shown) to confirm which is the L-lactate peak. The peak at retention time 3.9 minutes corresponded to lactate. It is important to note that the overlaid graphs are increasing concentrations of lactate solution (the ones we prepared for standard curve plotting) and not spiking.
Standard Graph with Error Bar
This standard curve of L-lactate plotted using the areas under the peak of 5 different concentrations helps us find out the concentration of HT-29 cell’s spent media. We obtained the value from literature to be ~18 mM and hence we chose concentrations for the curve ranging from 10 mM to 100 mM.
Overlaid chromatogram of spent media and spiking (24 hours-HT-29)
This graph is obtained by injecting the spent media of a 24-hour culture of HT-29 grown in a 60 mm dish. A peak is obtained with a similar retention time of that of the standard L-lactate solutions, which indicates that the media has L-lactate in it. To confirm that the peak is solely due to L-lactate, we injected increasing concentrations of L-lactate along with the media (spiking) and a corresponding increase of area under the peak was observed. Thus, we can conclude that the peak obtained from spent media is that of L-lactate.
Biological triplicates of this experiment yielded lactate peaks with different areas which when interpolated on the standard curve gave us an average value of 16 mM lactate.
Overlaid chromatogram of RPMI media and spent media (24 hours)
The purple line is the curve obtained by injecting fresh RPMI media with 10% FBS.No significant peak was observed at lactate’s retention time (3.9 mins). The overlaid black line is the readout from spent media showing a visible lactate peak.
Characterising the sfGFP Constructs
The regulatory element lldR binds to lldRO1 and lldRO2 (operator regions of lldPRD operon) 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 instead lldR complex with L-Lactate remains bound to LldRO1 acting as a transcriptional activator.
lldRO1-J23117-lldRO2 was characterized using sfGFP as the reporter gene under the control of RBS of different strengths, namely, Strong, Medium and Weak(Part:BBa_K3110040, Part:BBa_K3110041, Part:BBa_K3110042).
To test this part we have designed two plasmids:
O1PO2- RBS-sfGFP-terminator in a medium copy plasmid pSB1C3
P-RBS-sfGFP-terminator in another pSB1C3 (Same Promoter without the operator regions) - as a control
On adding lactate to the culture, L-lactate binds to lldR thus leading to enhanced expression of sfGFP only when O1PO2 is upstream of sfGFP.
Graph 1
As can be seen in the graph 1, when P-strongRBS-sfGFP is there, the fluorescence level is not varying much with an increase in L-lactate concentration. But, in the case of O1PO2-strongRBS-sfGFP, the increase in lactate concentration has lead to an increase in the fluorescence output.
Graph 2
In the case of weak RBS however, a clear increase wasn’t observed (Graph 2) and hence strong RBS is an optimum construct to use.
As the lactate concentration is increasing, the amount of free lldR (L-Lactate Repressor) inside the cell is decreasing as well as the lldR bound to lactate (activator) is increasing - so, the rate of sfGFP expression is also increasing with increasing Lactate (up to a certain concentration). After lldR is saturated with L-lactate, the sfGFP expression becomes steady. The lactate concentration of around 20 mM has however provided an unexpected decrease in normalized Fluorescence and further experimentation is required to validate the reasons for the same.
The construct with strong RBS gives a clear sigmoidal pattern for detection of lactate with a threshold of around 0.45 mM. This threshold doesn't help us since the construct will fail to distinguish Cancerous vs Non-cancerous microenvironments, both of which have lactate concentration higher than 0.45 mM.
Hence we have intended to use this construct in combination with our library of four gene constructs: lldD, lldP+lldD, lldR+lldD and lldP+lldR+lldD under promoter and RBS of different strengths.
Graph 3
We have characterized the new part Medium Promoter Strong RBS lldD which was cloned into pSB3K3 (a low-copy number plasmid) which is compatible with pSB1C3. This plasmid along with lldRO1-J23117-lldRO2 Strong RBS sfGFP (which was cloned into pSB3K3) was co-transformed into BL21 (DE3) and the assay was carried out. lldD (L-Lactate Dehydrogenase) cleaves L-lactate to Pyruvate thus depleting the amount of lactate in the cell.
This, we thought, would be the best way to control the expression threshold of lldRO1-J23117-lldRO2 (Graph 3).
Graph 4
Ectopic expression of lldD should lead to lower levels of Lactate in the cell which in turn would lead to higher levels of free lldR for transcription inactivation. And a higher dose of lactate would be required to turn on the expression. And indeed this is what we observed (Graph 4). We observed an increase in threshold from 0.45 mM (without lldD) to 2 mM (with lldD). Although this won’t be able to differentiate cancer from non-cancerous cells, the use of a strong promoter upstream to lldD and an appropriate combination of lldR and lldP in addition to lldD should. However, an increase of threshold suggests that our idea of using lldD as the modulator will work. A major drawback that we observed was a decrease in sensitivity i.e. even at sub-threshold lactate levels, the fluorescence observed in the presence of lldD was high. Not only this, at the aforementioned threshold levels, the intensity was lower than in the case without lldD. This could be rectified by using lldP and lldR along with lldD.
YebF IL12 FLAG Secretion
For both the YebF IL12 FLAG constructs, we wanted to see if the protein was being produced. To check for protein production, we cultured BL21 (DE3) transformed with the plasmids and collected both the pellet and supernatant of the overnight culture. For the constitutive promoter construct, the control was a culture of non-transformed BL21 (WT).In case of the lactate inducible promoter construct, a 1 mM lactate induction was given and this induced culture was compared to an uninduced one.
The gel images below show no visible band difference between the pellet and supernatant fractions of the samples. This indicates that the protein itself was not produced.
In order to verify the production of IL12, we performed a western blot with the inducible O1PO2-Strong RBS -YebF IL12 FLAG-Terminator construct. We induced BL21 carrying this construct with 1 mM lactate and had an uninduced control to compare to. Since our protein (74.04 kDa) has a FLAG tag, we probed the membrane with an anti-FLAG antibody (raised in mouse) and then imaged using a secondary anti-mouse HrP conjugated antibody. We have also loaded a positive control to ensure that the antibody worked fine.
The blot image shown below was obtained after a 10 minute exposure. The positive control shows the expected size (~36 kDa) while our samples show some non-specific bands (~35kDa).
We thus concluded that the bacteria did not produce the YebF IL12 FLAG protein. We suspected that this is happening due to SOEing errors. Indeed, we confirmed this possibility after obtaining the sequencing results. However, we could not repeat the experiment with a new clone due to time constraints.
"We play with bacteria and sometimes they play us"
Contact us
Email: igemiisertirupati@gmail.com
Address:
Indian Institute of Science Education and Research, Tirupati
C/o Sree Rama Engineering College (Transit Campus),
Rami Reddy Nagar, Karakambadi Road,
Mangalam (P.O.) Tirupati -517507.
Andhra Pradesh, INDIA
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