Human Practices

Overlap Extension PCR was used to generate the fused CysK-T25, which was then ligated into a tetracycline resistant backbone (pCH450). Correct ligation was confirmed with restriction analysis. I-T18 was replicated from a larger genetic construct using standard PCR, and ligated into an ampicillin resistant backbone (pTrc99A). Ligation success was again checked with restriction analysis. To generate the background cell line for our engineered plasmids, a reporter GFP downstream of the chromosomal Pap operon was introduced using phage that had been grown in GFP-mutant-containing bacterial cell lines. The phage was then introduced to a CyA (Adenylate cyclase) KO strain to introduce the mutant GFP into the chromosome. The engineered plasmids were then transformed into the background cell line, which was then introduced to bacteria expressing the cognate toxin.

Pathogen Detection using an

EngineeredContact-Dependent Inhibition SystemJohn Errico, Zachary Haynes, Travis Smith, Hiro Sparks, Katie Lee, Sarah Lensch,

Andrew Ballin, Daniel Reinhart, Colton Bracken, & Tsuyoshi KohlgruberGraduate Advisors: Christina Beck & Dan Nguyen Faculty Advisors: Professor David Low & Professor

Omar Saleh Motivation & StrategyIntroduction

Santa Barbara County Science Fair

We hosted a discussion forum for middle school and high school students and hopefully got them to think critically not only about the field of synthetic biology but also the ethics of such research in the context of modern society.

NanoDays “I Nano” We partnered

with the Santa Barbara Museum of Natural History and introduced a new station at NanoDays focusing on DNA. While children extracted DNA from strawberries and built double-helical DNA models out of candy, parents were able to ask us any questions they may have had regarding synthetic biology.

University ofCalifornia

Santa Barbara

Contact Dependent Inhibition (CDI) is a bacterial defense and communication strategy employed by many strains of bacteria to inhibit the growth of different strains of bacteria upon contact.

When one bacteria contacts another, a toxin (CT) is inserted from the attacking bacteria into the recipient bacteria from the tip of the CdiA protein. Once inside the recipient bacteria, the CT either already displays activity or must interact with an endogenous permissive factor (e.g. CysK) to become active. Once active, the CT may act as a protease, DNase, RNase, or other class of enzyme to degrade cellular machinery resulting in the death of the recipient cell. Cell death only occurs when CT is inserted into a non-self strain of bacteria. When inserted into the same strain as the attacking bacterium (self-attack), CT interacts with an immunity protein, coded for downstream of the same strains toxin, which binds CT and inhibits it’s function. It has also been found that both CdiI and permissive factors may interact with CT simultaneously to form a ternary complex.

Ruhe, Low, & Hayes. Trends in Microbiology (2013)

Inspired by the CDI system, can we engineer its components so as not to kill a cell but to trigger the expression of a particular gene? In other words…

We aimed to design a system where insertion of a specific CT into the recipient cell triggers transcription of a gene in that cell. Our "attacker" E. coli strain expresses a specific CdiA

on its cell surface that delivers a selected CT to our "indicator" strain.

Our “indicator” E. coli strain has the permissive factor CysK as well as the CT’s cognate immunity protein to negate the toxic effect of the delivered CT…both bind to the delivered CT.

By fusing functional parts to CysK and cdiI, we can trigger these parts to interact only when the CT is present. Adapting what is done in commercial bacterial two-hybrid systems – where two halves (termed T25 and T18) of the adenylate cyclase enzyme are brought into close proximity, enables formation of fully functional adenylate cyclase – we fused CysK to the N-terminus of T25 and the cdII to the C-terminus of T18. The functional adenylate cyclase then uses ATP to generate cyclic AMP (cAMP).

With GFP under the control of a pPap promoter, which is cAMP-dependent, GFP will only be produced when CysK-T25 and cdiI-T18 both bind to the delivered CT…which is only present in the “indicator” strain upon contact and delivery from the “attacker” strain. In the future, genes other than GFP may be inserted under the control of the Pap operon. We think that genes leading to the production of antibiotics would be very exciting. Antibiotic products would only be produced upon contact with a specific strain, thereby enabling strain-specific antibiotic delivery.

Can we develop a system of contact- dependent gene expression?

Methods & Results

1.8Kbp = CysK-T25

Engineering a bacteria that can induce gene synthesis based on extracellular contact with non-self bacteria proved to be no easy task. Creating a CysK fusion plasmid using overlapping-extension PCR and taking that same PCR product all the way through to transformation was unsuccessful the first four times it was attempted. Additionally, extracting and cloning I-T18 had it’s own set of problems. Mainly, contamination at the transformation stage resulted in incorrect plasmid transfection that showed up as an incorrect weight drop-out on restriction analysis. These challenges were overcome eventually, but at a cost of a significant amount of time that resulted in our inability to test the model we had created. The beauty of this system is it’s customizability. Different immunities can be incorporated to provide reporting on more than one strain. The gene product can be changed so that contact induces synthesis of whatever the engineer designs. Even the secondary messenger produced can be slightly varied. It will be up to future iGEM teams, UCSB-based or otherwise, to take this project to completion and even further beyond.

DiscussionFaint 1Kbp = I-T18