CONTRIBUTION
OVERVIEW
When we were brainstorming ideas for our project, we really wanted something that could be of use to many iGEM teams to come. We found it hard to find anything that made it easy to detect pathogens, biomarkers, or just arbitrary protein epitopes. When we were resaerching hwo to solve this problem we came across a recent breakthrough in protein design. The Baker Lab had created a modular biosensor. As we read the ltierature, we realised that if we could create a complete guide to creating a biosensor that it would be incredibly useful for future igem teams. That is exactly what we went on to do. Firstly, we will explain our entry for BEST NEW COMPOSITE PART and following on from that we will display the different protocols needed to use the basic part. Altogether this will provide a detailed guide for any iGEM team to create a biosensor for any epitope!
FIRST CONTRIBUTION: BEST NEW COMPOSITE PART
The Baker Lab created a state-of-the-art modular biosensor [1]. We decided, with permission from the Alfredo Quijsno-Rubio, that this modular biosensor should be introduced to iGEM. The biosensor consists of a Cage (LucCage Part Number: BBa_K4932002) and a Key (LucKey Part Number: BBa_K4932003) which make up the composite part (Modular Biosensor Part Number: BBa_K4932004).
HOW DOES THE BIOSENSOR WORK?
Please note the science behind our project is addressed in most detail within the engineering section of this webiste. However, here we will give a brief overview.
There are two parts to the biosensor system: The cage (LucCage) and the key (LucKey). LucCage contains a 'target binding motif' which is designed to specifically target an epitope of your choice (e.g. the baker lab used these sensor to target the covid spike protein). LucCage also contains a latch and a sequestered portion of a luciferase (provides luminescence when complete). When the cage binds to the target of interest, the latch opens, and the cage is said to be in the open configuration. As seen in the diagram, when the cage is in the open conformation, the key can bind. When the key binds, the key provides the rest of the luciferase protein meaning a luminescence signal is given off. This indicated a positive test for the target region.
WHAT WAS OUR 'TARGET BINDING MOFIT' ON THE MODULAR BIOSENSOR?
When using the modular biosensor, future iGEM teams will have to decide on an epitope of their choice to use. In the Baker Lab paper there have been many different proteins targets tested. These include using the spike protein as the 'target binding motif'. This results in the sensor detecting COVID anitbodies which in the pandemic proved incredibly hard to do. Other examples include using other natural troponins to detect cardic troponin I in myocardial infarction diagnosis. This is ingenious because it relies on the natural binding ability of troponin. We would advise that future iGEM teams using the mdoular biosensor try to use other proteins found in nature.
We used bacteriocins, as seen in the picture above. Bacteriocins are a kind of ribosomal synthesized antimicrobial peptides produced by bacteria, which can kill or inhibit bacterial strains closely-related or non-related to produced bacteria, but will not harm the bacteria themselves by specific immunity proteins [2]. This means we can use proteins evolved by bacteria themselves, to detect bacteria of a certain type. This could be a whole new class of diagnostics, not just using bacteriocins but using proteins that are similar in function found in all sorts of pathogens and orgnaisms. There were some interesting design considerations when we did this for example we had to flip the whole sequence because bacteriocins are encoded backwards as compared with regular proteins. This meant we had to encode our whole Cage and Key proteins backwards which we did not think would be feasible!
SECOND CONTRIBUTION: A COMPLETE GUIDE TO MAKING A BIOSENSOR
It will be useful to show teams that this modular biosensor works and it will be interesting to see where people can take this new method for creating biosensors. We are sure that other iGEM teams will have incredibly ingenious ideas of how to take advantage of this modular biosensor. For that reason we thougth we would put together a step by step guide in how to do this. It took us hours and hours to research and test the best protocols and so instead of letter all of that time go to waste we decided to put that in to a detailed guide.
PLASMID MAPS:
The Baker lab did not provide sequences of their plasmids. We requested plasmids from them and sequenced these ourselves. We then found out the length of the fragment they inserted and checked the insertion sites. Whilst doing this we also created our own plasmid using our own backbone and inserted our own gene in to this. The ones we have provided below are the sequence results of the plasmids we ordered directly from the Baker lab. We have also provided the sequences below.
Files:
Baker Lab Cage PlasmidBaker Lab Key Plasmid
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[1] Quijano-Rubio, A., Yeh, HW., Park, J. et al. De novo design of modular and tunable protein biosensors. Nature 591, 482–487 (2021).
[2] Yang SC, Lin CH, Sung CT, Fang JY. Antibacterial activities of bacteriocins: application in foods and pharmaceuticals. Front Microbiol. 2014 May 26;5:241. doi: 10.3389/fmicb.2014.00241. Erratum in: Front Microbiol. 2014;5:683. PMID: 24904554; PMCID: PMC4033612.