Team:IISER Tirupati/Parts
Parts
As you go through our parts, keep in mind that S, M, W stand for Strong, Medium and Weak; P stands for promoter and R is Ribosome Binding Site (RBS).
This is a library of the constructs that we planned on characterising. However, due to time constraints and technical difficulties, we could not characterise all of them. Here's a breakdown of the constructs:
- The first few constructs involve lldD, L-Lactate dehydrogenase, under promoter and RBS of varying strengths. It cleaves L-lactate to Pyruvate, and therefore, we used this part to tune the amount of lactate entering our cells. Therefore, lldD helped us establish a threshold of lactate concentration above which the cells will be able to detect lactate. Out of the parts that we planned for lldD, MP SR lldD worked for us and its our silver medal part!
- The next few constructs have L-lactate Dehydrogenase (lldD) and L-Lactate Permease (lldP) under the control of tunable promoters and RBS. lldD cleaves L-Lactate to pyruvate and lldP permits the entry of L-Lactate inside the bacterial cell. Our intention of using these part was to again have control over the detection threshold of L-lactate by the lldPRD regulatory region. Due to troubles in SOEing, we weren't able to characterise all of them.
- Next constructs have L-Lactate Permease (lldP), Regulatory element (lldR) and L-lactate Dehydrogenase (lldD) under the control of varying strengths of promoters and RBS which helps us tune their expression both at transcriptional and translational levels. lldP permits the entry of L-Lactate inside the bacterial cell, lldR has the dual role of an activator and a repressor and lldD cleaves L-Lactate to pyruvate. Again, technical difficulties with SOEing prevented us from characterising them.
- lldRO1 (or simply O1) and lldRO2 (or simply O2) are the operator regions which bind to the regulatory element lldR and inhibit transcription. lldR generally represses the Lactate Operon, possibly by forming a dimer, blocking the RNA polymerase from binding to the promoter. Upon binding of L-Lactate to lldR, 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 our desired output (activation at a lactate threshold similar to cancer microenvironment lactate concentration). Out of these, we were able to successfully characterise O1PO2 WR sfGFP, O1PO2 SR sfGFP, Constitutive P WR sfGFP (in our case the output is super-folded GFP or sfGFP)- and these are our bronze medal parts.
- We then have FimH protein with a mutation that leads to substitution of 1 amino acid at the 136th position, disrupting its ability to bind to mannose. Introduction of the above mutation and adding RPMrel, a colon cancer homing peptide downstream ensures it selective binding to colon cancer cells. This part was already developed with an arabinose promoter, a SpyTag and a HisTag downstream to RPMrel. We amplified the FimHRPMrel from this existing part, and to this, we ligated a strong constitutive promoter and strong RBS for greater expression. While the SOEing was successful, we failed to get any positive clones.
- Lastly, YebF is a 13kDa protein that is known to be secreted into the extracellular medium by E. coli strains used in the lab. This particular protein was used with IL-12 in our design, in order to mediate the secretion of the cytokine from the bacteria. The part was used with both constitutive and inducible promoters, but the results were inconclusive. Western blots were done with both the cell pellet and concentrated supernatant but the protein was not detected. Possible reasons for failure could be because of SOEing errors in the promoter and RBS regions.
References:- Aguilera, L., Campos, E., Gimenez, R., Badia, J., Aguilar, J., & Baldoma, L. (2008). Dual Role of LldR in Regulation of the lldPRD Operon, Involved in L-Lactate Metabolism in Escherichia coli. Journal of Bacteriology, 190(8), 2997–3005. doi:10.1128/jb.02013-07
- Zhang, G., Brokx, S., & Weiner, J. H. (2005). Extracellular accumulation of recombinant proteins fused to the carrier protein YebF in Escherichia coli. Nature Biotechnology, 24(1), 100–104. doi:10.1038/nbt1174
"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
Follow us on:
Our sponsors
