Team:IISER Bhopal/Introduction

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Introduction

Our project this year is aimed at developing a cold-tolerant cloning/expression system for the stable expression of recombinant genes/proteins in mesophilic hosts grown under suboptimal conditions.

Did you tune out already? Worry not! Sit back, and let our garrulous smooth talkers spin a fascinating yarn to keep you captivated. After all, iGEM’s all about learning the fun way!

Let’s begin with the basics. Mesophilic organisms are your regular next-door microbes which grow optimally in moderate temperatures (usually defined between a fairly nippy 20°C to and a decidedly uncomfortable 45°C). Their more adventurous cousins are the extremophiles - the legends who not only survive but outright thrive, in the face of some of Earth’s harshest environments. Be it corrosive pH ranges, crushing pressures, near-melting temperatures, tons of radiation, or (of particular interest to us) freezing cold conditions, these wily minions have well and truly proven their hardiness.

Fig.1.1: Comparison of growth of mesophilic bacteria at different temperatures.

Fig.1.2: Imaginary depiction of extremophiles.

However, despite their eyebrow-raising attributes, the fact remains that such microbes are still freaks of nature as far as a significant proportion of research is concerned. Detailed mechanisms enabling their optimal functioning under such inhospitable conditions are yet to be satisfactorily elucidated, and it’ll undoubtedly be quite a while before we can firmly gain a handle on understanding and appreciating their biology.

By comparison, decades of research on some of the most classic mesophilic systems (think E. coli !) have ensured that researchers can confidently claim a wealth of knowledge related to their functioning, regulation, and characteristics. This is an especially important edge when it comes to direct industrial application of such microbial systems for the production of recombinant proteins and compounds. The reasons for this are fairly obvious - the more complete our knowledge about a biological system, the easier (and more reliable) become our efforts to optimize its performance as per our specifications. Think of this like chess - the more familiar and experienced you become with the game, the easier you find it to crack opponent strategies and devise increasingly clever (and sometimes complex) gameplans.

However, there is a particular class of proteins which beats regular mesophilic systems (Actually, there are quite a few proteins which are not suitable for expression in mesophilic systems using standard protocols. Here, we are focusing on only a particular class among them). These are called psychrophilic enzymes, and they are a very special class of proteins expressed only by extremophile bacteria that inhabit cold temperature regions. We’ll be discussing these at length under the Project Inspiration section, including why they are so very important (not to mention fascinating) from a biotechnological point of view. But for now, suffice it to say that the stable expression of these in conventional mesophilic hosts is a pretty hard nut to crack.

Which isn’t really hard to guess. Think about it - if you were an Eskimo, you most certainly would feel out of place in the middle of the Sahara! Psychrophilic enzymes face very similar issues when forced to express in mesophilic host systems. The so-called “regular” temperatures of growth, say around 37°C, is a far cry from their native conditions which border around 0°C. And if you thought the simple way out was to instead place the mesophilic hosts at 0°C, well, that ain’t happening anytime soon. Most mesophilic systems will either outright give up their ghosts under such bone-chilling conditions, or grow so inefficiently that no industrial outfit would ever consider them worth the trouble..

Clearly, what’s required is not just a marriage between two systems poles apart from each other, but a hybridisation which combines the best of both worlds. After all, if it's targeted at the industry, efficiency had better be the watchword.

Hence we’ve decided to focus on engineering systems which can combine the survival ability of cold-tolerant extremophiles with the diverse advantages offered by traditional mesophilic systems, and thereby enable a crossover between the two.

And that’s exactly why we decided to call our project ELSA - E. coli Learns Suboptimal Adaptation!

Thus, we primarily plan to use cold-tolerant genes to develop an expression system for the synthesis of psychrophilic enzymes and enhance their solubility at low temperature.

So before we move onto the Project Design, let’s first take a delightful tour through the Project Description section, wherein we’ll tell you the lovely story of how ELSA came to be!

@iGEM IISERB