1. Daniel Cui & Eileen Liang
Bacterial Computing
Daniel Cui & Eileen Liang

2. Overview Solving the Hamiltonian Path Problem
Diagnosing stomach cancer (design)

3. Hamiltonian Path Problem
HPP: whether there is a route in directed graph from beginning node to ending node in such a way that each vertex is only visited once
Bacterial computers: programmed by constructing gene circuits to execute algorithm that is responsive to environment and result can be observed

4. Advantages of Using Bacterial Computing
Are autonomous
Can adapt to changing conditions/evolve to meet challenges of problem,
Exponential growth of bacteria continuously increases the number of processors working on the problem

5. Abstractions DNA segments: were treated as edges of directed graph
Treated all nodes, except for terminal one, as genes split into two halves
Arrangement of DNA edges: represented HPP solution and displayed new phenotype

6. How Bacteria were Programmed/Designed
Used E coli and programmed it by constructing gene circuits to execute an algorithm that is sensitive to its environment and result can be observed
Researchers: encoded three node directed graph as DNA segments that were autonomously shuffled randomly inside bacteria with recombination system
Recombination system: stimulated by Hin recombinase which inverts DNA fragments between hixC sites

7. Mathematical Modelling
Determining the effects of orientation and order of DNA on probabilities of detecting a solution to the HPP
Using permutations, matrices and probability
Determining the number of bacteria necessary in order to assure a definite plasmid with the solution to the HPP
1 billion identical, evenly distributed plasmids in order to be 99.9% assured of a result

8. Proof-of-Concept Experiment
Use directed graph with three nodes and three edges
Unique HPP pathway: starts at RFP node and travels via edge A to GFP node and from GFP to TT via edge B
Edge C (detractor): from RFP to TT node which is the ending

9. DNA Constructs
Used to encode solved HPP solution as positive control and two unsolved configurations
Solution: must start at RFP node and end at GFP node
DNA edge A: starts at 5 half of RFP and ends at 3 end of GFP
DNA edge B: starts at 3 half of GFP and ends at double terminator TT
DNA edge C: starts at 3 half of RFP and ends at double terminator TT

10. Expression Cassettes Contain all three DNA edges
Designed expression cassette to contain all three DNA edges (no recombination yet)
Ensure solutions begin at RFP node : cassette starts with bacteriophage T7 RNA polymerase promoter, RBS, and a 5 RFP prior to first hixC site
RBS
5 RFP
hixC

11. DNA Constructs Used in Experiment
ABC and ABC’ (backwards edge C): two solutions and both expected to fluoresce yellow because RFP and GFP genes are both intact
ACB and BAC
ACB: RFP intact but GFP is not which makes it fluoresce red
BAC: both genes are not intact which makes it not fluoresce

12. Bacterial Cotransformation
Helped produce random ordering of edges in directed graph
Co transformation: 1. Plasmid conferring ampicillin resistance and containing one of three constructs & 2. Plasmid encoding tetracycline resistance with a Hin recombinase expression cassette
Resulting co transformed colonies: grown for isolation of plasmids containing Hin-exposed HPP constructs, and isolated plasmids go through 2nd round of transformation into bacteria that expressed bacteriophage T7 RNA polymerase

13. Results
Before recombination: three constructs which consist of ABC (yellow), ACB (red), and BAC (no fluorescence)
Recombination: produced random orderings of edges in directed graph using each starting construct

14. Design Use Bacteroides thetaiotaomicron to diagnose stomach cancer
Bacterial computing: enable bacteria to sense the presence of abnormal growth of stomach cells
Will fluoresce green in solid waste that has been excreted by the body to notify the patient

15. What is Stomach Cancer?
Is a type of cancer that originates in the mucus-producing cells on inside lining of the stomach (adenocarcinoma)
Begins when cell receives a mutation in its DNA and starts to divide at a rapid rate, enabling it to form a tumor and to eventually metastasize

16. Helicobacter pylori
Bacterium that lives in the mucus lining of the stomach and converts urea to ammonia → acids from the stomach enters the lining and causes ulcers
Commonly linked with non-cardia gastric cancer
Those with H. pylori infections are 8 times more likely to develop non-cardia stomach cancer

17. Diagnosis Using Bacterial Computing
Bacteroides (engineered to produce GFP): will utilize quorum sensing to sense growth of Helicobacter pylori
Presence of H. pylori: will elicit response in Bacteroides, causing it to activate its gene for fluorescence

18. Diagram
Truth Table
Autoinducer-2
GFP Promoter 1

19. Alternate Methods
Endoscopies and Endoscopic ultrasounds, in which probes are inserted through the mouth and used to check for the presence or spread of cancer. If concern is raised, biopsies can be performed to yield a definite result
However these methods are not always useful for early detection
MIT: used engineered bacteria that can penetrate and grow in the tumor’s environment in order to express gene for lacZ enzyme which acts on galactose that is linked to luciferin (already injected into patient)

20. Sources http://www.pnas.org/content/97/4/1328/F1.large.jpg
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