Goal: Demonstrate cell-to-cell communication.
I love the idea of building a “computer” using cells. Unfortunately, you can only fit a handful of logic gates into a single cell. If you want to build a circuit with hundreds or thousands of gates, you need to split them up across multiple cells, so you need a way to send signals from one cell to another.
I want to focus on that cell-to-cell signalling for my final project.
This project is based on the Ph.D. thesis https://dspace.mit.edu/handle/1721.1/152844. He implemented a reduced version of the MD5 hash function in E. coli — that’s really cool! Cryptography has long been a passion of mine.
For cell-to-cell communication, you want one cell to secrete a signaling molecule, and another cell to detect it.
Padmakumar’s thesis identified some signalling molecules that work well. For this project, I want to try using two of them:
LuxI enzyme.CinI enzyme.These signalling molecules are nice because they can be produced by a single gene (easier to engineer), they’re not toxic to the cell, supposedly have good dynamic range, and these two molecules are not cross-reactive.
This cell expresses the LuxI enzyme which produces 3-oxo-C6-HSL.
In his thesis, Padmakumar created the sender devices manually, like this:
However, since we can order cloning vectors from Twist, I will just put the LuxI gene into the off-the-shelf pET-28b(+) expression vector for E. coli, which comes with the T7 promoter and RBS already built-in. I just need to supply the gene sequence.
https://www.uniprot.org/uniprotkb/P12747/entry
MTIMIKKSDFLAIPSEEYKGILSLRYQVFKQRLEWDLVVENNLESDEYDNSNAEYIYACDDTENVSGCWRLLPTTGDYMLKSVFPELLGQQSAPKDPNIVELSRFAVGKNSSKINNSASEITMKLFEAIYKHAVSQGITEYVTVTSTAIERFLKRIKVPCHRIGDKEIHVLGDTKSVVLSMPINEQFKKAVLN
(193 aa)
Plasmid design: