Team:IISER Bhopal/Description

iGEM IISER Bhopal

Description

Now that we’ve explained what E.L.S.A. is all about, if you haven’t read that, please do so hereit's time to talk about how E.L.S.A. came into being - specifically, our motivation and rationale for deciding upon this project for our 2019 iGEM entry.

At the very beginning of time (circa January ‘19), when our hostel rooms were still cold and the labs enticingly cosy, twenty younglings gathered to forge a confused alliance - which wasn’t so sure about who our lore’s lead villain would be. Over subsequent weeks and innumerable rounds of coffee, some very interesting ideas began to emerge. During these sessions, we came across a particularly interesting proposition - the stable expression of psychrophilic recombinant proteins under sub-optimal conditions. While this was something almost none of us had any prior knowledge about, we were all quite intrigued by the invitingly novel premise of such a problem.

Following extensive literature explorations, we summarized that:

  1. Psychrophilic enzymes (enzymes produced by bacteria growing at cold temperatures) were industrially relevant for a bevvy of applications ranging across fruit juice clarification, meat processing, production of special wines, and so on.
  2. Most industrial and laboratory processes are optimized for mesophilic hosts, simply because of the relative culturing ease and prevalence of the latter; as well as the substantial background of work that had been done on them which allowed for reliable characterization.
  3. The principal bottleneck was an inability to stably express these enzymes in mesophilic hosts, the frequent formation of inclusion bodies and difficulties in inducing mesophilic hosts to grow efficiently at sub-optimal temperatures.

These realizations were further tempered by the various discussions we had with our faculty advisors (who are all active researchers), and representatives from certain industries which deal with psychrophilic enzymes. Thereby, our major objectives became much clearer. We would have to work towards designing a system that:

  1. Allowed the easy, efficient cloning of different recombinant proteins, so that it could be truly modular (important to ensure broad-spectrum applicability)
  2. Enabled the stable growth of mesophilic hosts at sub-optimal temperatures.

Both these objectives could be achieved if we could design strategies allowing for the efficient folding of proteins at cold temperatures - essentially requiring a cold-tolerant chaperone. If a mesophilic host expressed such chaperones, then these would take care of protein folding processes even at sub-optimal temperatures, thereby allowing the transformed host to far outperforms the wild-type strains under such conditions. This would, in turn, allow the stable and efficient expression of our recombinant protein of interest, which would be active only at such low temperatures.

Fig.1 :Homology model TF of P. haloplanktis

Upon inspection of the E. coli system, we observed the presence of a ribosome-associated molecular chaperone, the trigger factor. The particular variant found in mesophilic systems like E. coli helps guide the initial folding steps of growing polypeptide chains. This component was a target of interest because a homolog- the Trigger Factor (TF), as isolated from the P. haloplanktis - had been reported as a highly active monomeric chaperone, which gives an optimal performance under cold temperatures. Since both proteins are homologs, we considered it a reasonable stretch to assume that the cloning of P. haloplanktis-derived TF into mesophilic system would help us achieve at least part of our aforementioned goals. (For a detailed elucidation of the TF, please refer to our “Basic Part” page).

These properties helped us zero-in on the P. haloplanktis trigger factor (hereafter referred to as TF), as a part of interest which could potentially confer cold tolerance to a mesophilic host. Following this, we searched for past iGEM teams who would’ve worked along similar lines. While no team has so far worked specifically on the TF, we found that Team Amsterdam from the 2011 edition had worked on a system involving the Cpn 10/60 (as isolated from Oleispira antarctica). The chaperonin 10/60 is homologous to the GroEL/ES chaperonin system in E. coli. Essentially, these are the active assistants for any protein folding processes. The advantage here was that Cpn 10/60 is a cold-active component, i.e., unlike the mesophilic chaperone systems which are highly inefficient at lower temperatures, these cold-active entities perform optimally at cold temperatures. Cpn 10/60 has a double ring structure, which predominantly retains a double ring form at mesophilic temperature ranges (excess of 24 °C), but shifts to a single ring form at lower temperatures (4°C-8°C). This is especially relevant since single ring conformations are more energetically efficient.

However, Team Amsterdam was unable to co-express Cpn 10/60 within a single construct. Being intrigued by this, we decided to not only attempt the same but also compare its performance with our proposed TF-inclusive construct. We soon realized that this would hardly be accurate - for one, they both have different temperature changes, on top of which TF is a co-chaperone, whereas Cpn10/60 is a primary chaperonin. We also came across a system developed by Agilent - the Arctic Express cell line, which involves a Cpn 10/60 recombinant protein expression in E. coli cells, over the conventional GroEL/ES. However, this system suffers from a handicap of attainable temperature ranges, as it experimentally performs best over a 10°C-12°C range, whereas the theoretical range is 4°C-12°C.

Taking all these into consideration, we decided to design a construct which would feature a TF insert, along with the Cpn 10/60 system, so as to achieve a larger global temperature range for the transformed chassis organism.

Fig.2Transition from double ring structure to single ring structure at low temperatures.