1. 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 Engineered
Contact-Dependent Inhibition System
John Errico, Zachary Haynes, Travis Smith, Hiro Sparks, Katie Lee, Sarah Lensch,
Andrew Ballin, Daniel Reinhart, Colton Bracken, & Tsuyoshi Kohlgruber
Graduate Advisors: Christina Beck & Dan Nguyen Faculty Advisors: Professor David Low & Professor Omar Saleh
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.
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?
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.
Faint 1Kbp = I-T18