CHE.496/2009/Responses/a10

Genetic circuit engineering (part 2)

• Discussion leader: Brandon

Patrick Gildea's Response

• Environmental Signal integration by a modular AND gate
  • This article describes the construction of a AND gate circuit in E. Coli based on parts from the registry. Figure 1 illustrates the construction of the promoters and repressor genes that form the AND gate. The ouput genes (gfp in this example) is only expressed when supD and t7ptag mRNA is present. If we wanted to design a system where we wanted to activate the expression of a gene when two inputs are present; this genetic circuit would be a suitable tool for that purpose. Furthermore, as a side project we could scale up this circuit to include more inputs. For example, a three or four input AND gate would make an interesting project where the modeling of the circuit would be required. A AND gate with more than 2 inputs would reduce the complexity of a system that couples multiple AND gates to form a large circuit that expresses a gene only when more than 3 or four inputs are required.
• Environmentally Controlled Invasion of Cancer Cells by Engineered Bacteria
  • The purpose of this paper is to describe a possible treatment for cancer using genetically engineered bacteria that can sense cancerous environments (via hypoxia detection) in a eukaryotic cell, then invade the tumor, and then destroy the tumor via invasion, a cytotoxic agent. Being able to direct bacterial organisms to target a certain environment is a wonderful tool in which these people took advantage of a promoter that operates in a hypoxia environment. It would be a good addition to the registry to build biobricks that can be used to selectively target environments in biological systems as opposed to something that just affects the whole system. Furthermore, of interest is how they were able to tune the input signal of the promoters to the output signal (invasion). Instead of using genetic circuits or regulatory networks or even the old and tried method of mutating promoters to get the desired promoter strength; the ribosomal binding sites were mutated to get the desired expression level in an attempt to reduce the translation rate of the output gene (invasion). Our project last year, attempted to control basal expression through transcription rate as opposed to translation rate. It would be interesting to learn which mode of control is better in different situations.
• Patrick Gildea 17:58, 22 March 2009 (EDT):

Rohini's Response

Environmental signal integration by a modular AND gate

• Microorganisms use genetic circuits to retrieve and process information from their environment
• synthetic AND gate made in E. coli bacteria from two promoters inputs and a activator output
• the AND gate has a modular design (reconnect the input and output signals to drive different cellular responses)
• the output of an AND gate is on when all of the inputs are on
• the AND gates have to integrate multiple signals in order to achieve sensing specificity

• Promoter inputs:

1) First promoter controls the transcription of T7 RNA polymerase gene by two internal amber stop codons blocking translation

2) Second promoter controls the amber suppressor tRNA supD

• Activator output:

-GFP (green fluorescent protein)

Problem-programming cells to identify an environment that is not naturally encountered and there is not a dominant signal

• How the AND gate works:

1) Input promoters respond to arabinose and salicylate

2) The first promoter transcribes mRNA encoding T7 RNA polymerase

3) The presence of SupD in the second promoter decodes the amber TAG stop codons as serine and translation occurs

4) When the T7 RNA polymerase gene is expressed this activate the output which expresses the fast-degrading green fluorescent protein

• Problem: the activity of the input promoter does not match the range required for the proper behavior of the circuit
• Solution: mutagenize the ribosome binding sites by rational substitutions or random mutagenesis. This will allow for the identification of functional rbss.
• Experiment-The researchers used natural input promoters that respond to magnesium and AI-1 and for the output, invasin gene. This would enable the bacteria to invade mammalian cells.
• Goal-incorporate genetic circuit design in systems with multiple sensors and actuators

Environmentally Controlled Invasion of Cancer Cells by Engineering Bacteria

• Bacteria have three functions:

1) Sense their environment 2) Distinguish between cell types 3) Deliver proteins to eukaryotic cells

• Purpose-engineer bacteria to sense the microenvironment of a tumor (hypoxic) and respond by invading cancer cells and delivering cytotoxic agents

Rohini Manaktala 20:19, 22 March 2009 (EDT)

Thaddeus's Response

Environmentally controlled invasion of cancer cells by engineered bacteria

• Purpose: To present the design of bacteria engineered to invade cancer cells.
• They introduced the gene for Invasin into E. Coli. This gene is what allows Yersinia pseudotuberculosis to invade mamallian cells expressing B1 integrins
• Allows E. Coli to invade tumor cells.
• Can also be placed under environmental control so that it only invades cells in a tumor like microclimate.
• Invasisn does not require any helper proteins.
• About 1-8% of cells were recovered in an assay in which invading cells were protected from antibiotics by mammalian cells which they had invaded.
• Initially invasion was independent of control because baseline transcription allowed invasion. RBS sequences were altered to prevent this.
• They tested every aspect of project alone in addition to testing final product.
• Invasin could be redesigned to bind new targets which would expand usage.
• This paper is useful because it expands on circuit design and brings to mind the importance of the strength of translation affecting systems which are meant to be controlled at the transcriptional level.

Environmental signal integration by a modular AND gate

• To describe the design and construction of an AND gate.
• AND gate helps identify specific environment by combining various environmental stimuli.
• More signals leads to more specificity.
• Only when both inducers are present does output function.
• They made the AND gate by putting stop codons in output of wone signal and make other output a stop codon repressor.
• Had to weaken RBSes again.
• The transfer function for the AND gate converts the strength of promotion to strength of output.
• Demonstrates that I1 is better for a leaky promoter because can be compensated for by I2
• Modularity was demonstrated by replacing inputs and outputs and maintaining the functionality of the circuit.

Thaddeus Webb 23:12, 22 March 2009 (EDT)

Maria

“Environmentally Controlled Invasion of Cancer Cells by Engineered Bacteria”

• bacteria controlled by environmental signals
• vibrio fischeri lux quorum sensing circuit
• hypoxia-response fdhF promoter
• or arabinose-inducible arcBAD promoter

• existing “output interfaces:”
  • biofilm production
  • population control (link quorum sensing to cell death)
• this example uses invasion to adhere and invade mammalian cells
• stimulates Rac-1, stimulates uptake (by attaching to beta-1 integrins)
• cell density
• hypoxia
• inducible inputs
• inv is under control of the quorum sensing lux operon- induced by anaerobic ,and is a fdhF promoter ot araBAD

• had to connect trigger to invasion output
• identified clones with inducible phenotypes from gene libraries
• inv gene controlled by tet promoter
• also:
• invasion gene controlled by araBAD –araBAD promoter and AraC gene replace tet promoter
• (arabinose dependent activation and looping-mediated

repression by AraC)

• arabinose is not metabolized, so promoter is used for over expression of foreign proteins
• worked at single copy

“Environmental signal integration by a modular AND gate”

• promoter 1: controls T7 RNA polymerase transcription transcription (2 stop codons blocking translation)
• promoter 2: controls tRNA supD suppressor
• respond to Mg2+ and AI-1: response : invade mammalian cell
• both inputs must be on to activate output
• if output is promoter, it can be linked to other circuits

• Maria Fini 17:30, 23 March 2009 (EDT):

Joe's Response

Environmental signal integration by a modular AND gate

• A synthetic AND gate in the bacterium Escherichia coli has been constructed that integrates information from two promoters as inputs and activates a promoter output only when both input promoters are transcriptionally active.
• The AND gate has a modular design with input and output promoters
• First promoter controls T7 RNA polymerase and second promotor controls tRNA supD
• The output florescent GFP gene is expressed only when supD and T7ptag mRNA is present.
• AND gate identifies specific environment utilizing various inputs (more inputs enhance specificity).
• Ribosome binding sites were mutated
• The input promoters responded to magnesium and Al-1, and the output to invasin. -- > so now bacteria can invade mammalian cells.

Environmentally Controlled Invasion of Cancer Cells by Engineered Bacteria

• Purposes of bacteria:
  • sense environment
  • distinguish between cell types
  • deliver proteins to eukaryotic cells.
• Invasin gene was introduced into E. Coli, so as in the previous paper, the bacteria can now bind to and invade mammalian cells
• Ribosome binding sequences were altered to enable control of the invasion; otherwise, normal transcription was all that was needed for invasion to occur.
• Target idea was to design bacteria to sense a hypoxic environment and then invade and destroy cancerous cells.
• Good example of synthetic biology-utilizing what was known to create and modify a genetic system to accomplish a purpose.

Joe Bozzay 19:05, 23 March 2009 (EDT)