20.109(S09)/PinkWF

=20.109 Research Proposal for Derek Ju and Alvin Chen=

Project Overview
Our goal is to rewire a bacterium to "miniprep" itself, when given some sort of an external stimuli (such as sensing of dense cell growth). This project will serve the following purposes:


 * Make the research process easier, cheaper, and more efficient for scientists working with bacteria.
 * Most laboratory strains have been genetically modified for scientific use; however our bacteria would be the first to be modified to do an experiment on itself.
 * Self-lysing of cells when dense cell growth is encountered - less chance for environmental contamination if genetically engineered bacteria escape from the lab
 * Serve as an example of a way synthetic biology can be used for cellular control
 * Be an application of using irreversible switches within genetic circuits
 * Be an example of sensors being built into bacteria

Background
Cell-cell signaling:
 * Need two devices: Sender device and Reciever device. Current devices are based on Lux system of V. Fischeri or analogs. http://partsregistry.org/cgi/partsdb/pgroup.cgi?pgroup=Signalling
 * Sender: BBa_F1610
 * Reciever: BBa_F2620 http://www.nature.com/nbt/journal/v26/n7/full/nbt1413.html. Characterization of this part by Drew Endy

Polymerases Per Second (PoPs)
 * Standard unit for signal via which information is transmitted in transcription-based logic devices. (http://openwetware.org/wiki/PoPS.
 * Common intracellular signal carrier for transcription-based devices. BBa_F2620 output is PoPs, so we'd have to have a trigger with PoPs input.
 * No in vivo technique for measuring PoPs directly, but can do it indirectly (look at endy paper above)
 * Abstraction hierarchy: http://partsregistry.org/cgi/htdocs/AbstractionHierarchy/index.cgi

Gene Gateway (need to figure this out)
 * Input would be an amount of PoPs generated from the cell quorum sensor
 * How do we set a threshold for PoPs toleration amount?
 * maybe simple constitutive inhibitor production until it's overwhelmed
 * Need to measure inhibitor production and how to calibrate this
 * Output leads to generation of lysozyme AND RNase (anything else)
 * IGEM 2004 polkadorks (http://parts.mit.edu/wiki/index.php/IAP2004:Polkadorks)

Biobricks
 * A lot of parts are available for us to use already
 * May need to add new parts (hopefully)
 * Need to characterize these parts
 * Not good for fusion proteins (do we need any?) but can use modified Silver standard for this

Leucine Zipper
 * Clump cells together (pros/cons to this?)

Reporters
 * attaching GFP, RFP etc to gene circuits would allow for quick, easy determination of whether it's working
 * Good for debugging
 * Should only be for experimental design, final product should not have this

Lysozyme Gene (which?)

RNase A Gene (which?)

How to isolate only DNA?
 * Can we easily isolate nucleotides since RNA isn't present anymore?
 * Some kind of universal proteolysis? How to get rid of residues? Addition of phenol/chlorofrom can dissolve and denature proteins
 * How to isolate plasmid DNA vs genomic DNA? Can we find a way to do both?
 * Maybe if you get rid of enough proteins/RNA then u can just use the mixture to do the next reaction (transformation, digestion) with some type of efficiency, but this would have to be studied
 * When bacteria are lysed under alkaline conditions both DNA and proteins are precipitated. Some scientists reduce the concentration of NaOH used to 0.1M in order to reduce the occurrence of ssDNA. After the addition of acetate-containing neutralization buffer the large and less supercoiled chromosomal DNA and proteins precipitate, but the small bacterial DNA plasmids can renature and stay in solution.

Characterization of parts
 * Efficiency of expression of lysosome and RNase
 * Efficiency of gateway
 * Efficacy of RNA degradation
 * Efficacy of transformations with this mixture OR purity of plasmid if we decide to purify

How does miniprep work?
 * What does each buffer, spin column, etc do?
 * How much does it cost?

How does genomic DNA isolation work??

Research Problems and Goals
Competing Technologies: Qiagen miniprep, Berkeley clonebots Safety: Don't know toxic effects of inserting these genes, but it should make it safer by having a self-aptosis mechanism

Project Details and Methods
In order to complete our project, we will attempt to incorporate several new genes into E. coli, which will encode for various proteins.


 * 1) We will insert a gene encoding for a binding protein that will be attached to the membrane of the bacteria. This protein will most likey contain leucine zippers and as a result the bacteria will lump together in culture.
 * 2) The attachment of the binding proteins to eachother will indicate clumping of cells, which will release a signal to toggle a "genetic switch". This switch will be a collection of genes, which when combined creatively, can have an input and output.
 * 3) The activation of the genetic switch will lead to expression of a gene encoding for lysozyme, which will lyse the cell.
 * 4) The activation of the genetic switch will lead to expression of a gene encoding for RNAse A, which will degrade the RNA in the cell.

Project Overview (abstract)


 * Cells secrete chemical --> cells detect high levels of extracellular chemical --> Activate gateway --> produce Lysozome & RNase

Gene Pathway (abstract)


 * Quorum signal sender device ---3OC6HSL---> Quorum signal reciever ---PoPs---> Genetic Gateway ---ON--->Lysozome & RNase production

Predicted Outcomes
Don't know:
 * secretion efficiency of lysosome needed to lyse cell
 * same for RNase to degrade RNA
 * How to calibrate the threshold for PoPs to activate gateway (usually we wait for cell growth to reach a certain density until we miniprep it)
 * How do the modifications affect the cell
 * How to isolate DNA, and further how to isolate genomic from plasmid (make sure this is not harder than using a miniprep kit)

Resources Needed

 * Appropriate DNA sequences for genes, signal sequences, etc
 * Standard plasmid for transformation into E. coli, such as pSK1A2
 * Some of the DNA for the genetic switches can be retrieved from the Biobricks foundation