Team:IISER Bhopal/Engineering

Engineering Success

Engineering Success


Engineering success for our team involved constant ideation, designing, adjustment and improvement until we reached the final product - iβeta, capable of tackling diabetes by producing beta cells in vivo.

Unfortunately, our campus was locked down due to COVID-19 for essentially the entirety of our project, meaning we didn’t have access to a wet lab. Instead, we’ve designed a detailed experimental workflow, consisting of four modules of wet lab work to validate our hypothesis and demonstrate our parts’ functionality, along with a fifth module to improve our parts and correct for potential failures, and three more to build on our work and develop a device competent for testing in the clinical trial stages.

Technical Design Improvements

  • Our initial plan was to express the three transcription factors on an operon, as separate ORFs. However, it was pointed out to us that we would be unable to ascertain the stoichiometric ratios of translation of our 3 factors which was for our device to operate, so we tweaked our expression system.

  • We switched to a polyprotein-based BioBrick instead, where all three of our factors were joined directly by linkers under a single operon. They entered the target cell as one, achieving a roughly 1:1:1 stoichiometric ratio while being delivered to the gut cell.
    In order to ensure the polyprotein was separated into 3 functional transcription factors, only in our cells of interest, we needed to find a protease specific to our target cells - crypt base columnar cells(CBCCs).

  • At first, we tried to find a protease specific to our target cells - CBCCs. A promising candidate called SENP1 appeared. It helps chop SUMO tags from their conjugated proteins. A proteome-wide screen found that it was enriched in our target cells. We thought we'd found the holy grail!
    But it turned out that SENP1 shared a cleavage site with multiple proteases, some of which were expressed in other cells as well. Another issue we'd overlooked was the fact that it leaves a 100 aa residue at the C-termini of our factors, which could possibly impact their function.

  • Our current system does not rely on a host protease. Instead, we have sneaked in our own protease and designed it such that it is only functional in our target cells. We have used beta-catenin, the famous Wnt effector, usually degraded in most cells except the ones with an active Wnt cascade (as is present in our target cells).
    We have fused a protease - Tobacco Etch Virus protease with beta-catenin which is active only in crypt cells. Our device has a multi advantage it is a protease that efficiently cleaves our transcription factors whilst maintaining cell-specificity, safety and efficacy of our system.

Following the engineering cycle of Design, Test, Improve and Research. We were able to design a delivery system which is safe, effective and capable of producing beta cells in-vivo. To bring it to culmination we have designed several modules explaining the exact steps that would take our project from an idea to wet lab work that will give the real results.

N O T E: Unless specified, all bacterial culture work will be performed at 37°C and 200 RPM.

Extracting the plasmid (pGGA) and checking for intact restriction sites

Determining the bacterial growth kinetics

Confirming antibiotic sensitivity (Chloramphenicol, Broth + Plate)

Studying competency wrt our chosen vector

Establishing continuous cell line
(+ Checking for contaminants)

Inducing and confirming T3SS expression (SDS-PAGE)

Obtaining and amplifying synthetically generated construct

Loading construct onto vector (pGGA) using Golden Gate assembly

Transformation into cloning host + Screening

Transformation into test and control host (E. coli K-12)

Inducing and confirming effector expression (SDS-PAGE)

Observing T3SS secretion into media (SDS-PAGE)

Visualising and quantifying binding and intimate contact (Microscopy + Bound Cell Counting)

Observing effector translocation and nuclear entry (Fractionation + Western Blotting)

Quantifying insulin output (ELISA)

Constructing and validating antibody display apparatus
(See Module 2)

Studying the impact of antibody display on binding dynamics
(See Module 3B)

Constructing and validating expression of kill switch
(See Module 2)

Studying kill switch functionality

Constructing and validating secretion-tagged reporters (See Module 2)

Studying the impact of improved linker on expression, folding and secretion

Constructing and validating effector with improved protease sensitivity (See Module 2)

Observing improved protease sensitivity