1Laboratorio di Informatica Biomedica, Dipartimento di Informatica e Sistemistica, Università degli Studi di Pavia 2Centro di Ingegneria Tissutale, Università degli Studi di Pavia

ENGINEERING E. coli TO MULTIPLEX AND DEMULTIPLEX SIGNALSLorenzo Pasotti1, Mattia Quattrocelli2, Daniela Galli2, Maria Gabriella Cusella De Angelis2, Paolo Magni1

MOTIVATION. Synthetic Biology is a new area of Bioengineering which aims at designing and building novel biological functions and systems. The idea is to consider functional DNA elements as single modules, which can be assembled in a genetic circuit, just like capacitors and diodes are assembled in electric circuits. In order to introduce abstraction and standardization in Synthetic Biology, BioBrick standard parts have been created: they are modules with a standard interface, which makes circuit building very easy. International Genetically Engineered Machine (iGEM) competition is an annual worldwide student competition and it is the largest Synthetic Biology conference in the world. Participants are asked to build original biological functions and to compete for a variety of awards of excellence showing their projects in November at iGEM Jamboree at MIT. iGEM promotes the creation of novel biological functions providing a library of several BioBrick parts to students, who are welcome to use them and also to make their own standard parts as well. This year, University of Pavia participates at iGEM competition for the first time: a multidisciplinary team, which mixes engineering and biology skills, has been formed and an original Synthetic Biology project has been built up over the summer. In particular, project aims at providing multiplexing and demultiplexing capabilities in E. coli bacterium, implementing a genetic multiplexer (mux) and demultiplexer (demux), two devices that have a remarkable importance in electronic and telecommunication systems.

How to implement genetic logic gates?

Interconnecting logic gates: genetic implementation for mux and demux

Final BioBrick composite parts for genetic mux and demux. The implemented devices should be "universal", i.e. they have to be able to recognize every molecule as input and to express every gene of interest as output. For this reason, input promoters and output protein generators are not part of the two devices, allowing a final user to re-use them assembling the desired input and output elements.

Mux and demux in digital electronics. Mux transfers one of its input signals into a single output channel, while demux transfers a single input signal into one of its output channels. Thus, these devices can be considered as controlled switches.

Only the simultaneous presence of luxI and luxR can turn Plux promoter on, thus implementing an AND gate. We also considered a similar system, the las system.

AND GATE OR GATE

Two copies of the same gene under the control of two independent promoters can mimic an OR gate. In this example we replicate GFP gene under Plux and Plas.

A repressible promoter can mimic a logic inverter: cI inhibits the activity of P� promoter, which is normally on.

NOT GATE

40X FITC 40X FITC

40X FITC

40X FITC

HSL 1�M

40X TRITC

HSL 1�M

40X TRITC

no HSL

FITC

CH0

CH1

SELOUT

SEL

IN OUT0

OUT1

Mux:

Demux:

Let PA, PB and PS be three generic promoters that can be ACTIVATED respectively by the three exogenous molecules A, B and S. Let also GOI be the generic gene of interest. A genetic mux with inputs A and B, selector Sand protein GOI as output can be implemented by the following gene network:

Let PI, and PS be two generic promoters that can be ACTIVATED respectively by the two exogenous molecules I and S. Let also GOI0 and GOI1 be two generic genes of interest. A genetic demux with input I, selector S and GOI0 and GOI1 proteins as outputs can be implemented by the following gene network:

Three standard parts for Mux:

Two standard parts for Demux:

On/off testing. Eight "test parts" were built to study the qualitative behavior of the two devices. These parts have been incorporated into E. coli TOP10 and cell fluorescence has been acquired at microscope. Fluorescence was considered as a Boolean signal: high (present) and low (absent).

FITC

no HSL

40X FITC 40X

Quantitative testing. Two of the "test parts" were also tested quantitatively: steady state fluorescence intensity in response to different concentrations of 3OC6HSL inductor was considered. After cell fluorescence acquisition, an image processing algorithm to compute relative fluorescence intensity was used.

Truth table validation. The tests we performed validated five rows of logic gates truth tables. The validated rows are green-highlighted.

Mathematical modeling. Two systems of coupled ODEs which describe mux and demux gene networks were defined and simulated. The ODEs were based on the law of mass action and Michaelis-Menten and Hill equations, while parameters were taken from literature. Here we report a simulation result for demux model, in which IPTG is the input, aTc is the selector, RFP and GFP are OUT0 and OUT1, respectively . In this example, we computed state variables evolution in response to an IPTG step, without aTc. According to demux truth table, when IN=1 and SEL=0, then OUT0=1 and OUT1=0. So, in our simulation, RFP must be expressed, while GFP must not.

University of Pavia iGEM 2008 Team

References

http://partsregistry.org/Main_Page - Registry of Standard Biological Parts contains the collection of all existing BioBrick standard parts, including our new parts. In this web site the complete list and description of parts is reported.

http://2008.igem.org/Main_Page - 2008 iGEM competition web page.

http://2008.igem.org/Team:UNIPV-Pavia – University of Pavia iGEM 2008 Team wiki. In this web site the complete documentation of the project is reported.

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Conclusions• Thanks to iGEM competition, Synthetic Biology has been introduced into the University of Pavia for the first time.

• A multidisciplinary team has been successfully formed to work over the summer on two novel biological functions and to compete in iGEM 2008.

•The circuits implementing the two functions have been successfully assembled using BioBrick standard assembly, submitted to the Registry and ten tests havebeen performed to validate their behavior.

• After our 2008 summer experience, we look forward to working on Synthetic Biology again and to improving our skills in this captivating research field!

tetR is constitutively produced lacI is constitutively produced There is only a leakage production of cI because aTc

is not present

luxI is produced because IPTG is present

luxR is produced because cIis not present

lasI is produced because IPTG is present

There is only a leakage production of lasR because

aTc is not present

RFP is expressed because luxI and luxR are expressed, while

GFP is not, because lasI is produced, but lasR is not

CH0: A molecule

CH1: B molecule

SEL: S molecule

OUT: GOI protein

"logic 1" signal: presence of the element of interest (e.g.A, B, S, GOI)

"logic 0" signal: absence of the element of interest (e.g.A, B, S, GOI)

IN: I molecule

SEL: S molecule

OUT0: GOI0 protein

OUT1: GOI1 protein

"logic 1" signal: presence of the element of interest (e.g.I, S, GOI0, GOI1)

"logic 0" signal: absence of the element of interest (e.g.I, S, GOI0, GOI1)