Team:XMU-China/Project

Project Name

i-ccdB: intelligent Control of Cell Density in Bacteria

Project Description

We have developed a series of devices which program a bacteria population to maintain at different cell densities. We have designed and characterized the genetic circuit to establish a bacterial ‘population-control’ device in E. coli based on the well-known quorum-sensing system from Vibrio fischeri, which autonomously regulates the density of an E. coli population. The cell density however is influenced by the expression levels of a killer gene (ccdB) in our device. As such, we have successfully controlled the expression levels of ccdB by site-directed mutagenesis of a luxR promoter (lux pr) and error-prone PCR of gene luxR, and finally we have built a database for a series of mutation sites corresponding to different cell densities. An artificial neural network has then been built to model and predict the cell density of an E. coli population. This work can serve as a foundation for future advances involving fermentation industry and information processing.

Approach

3 Background

3.1 Quorum Sensing

Quorum sensing is a method of communication between bacteria that enables the coordination of group-based behavior based on population density. [3] It was first observed in Vibrio fischeri, a bioluminiscent bacterium that lives in the ocean. Bacteria that use quorum sensing constantly produce and secrete certain signaling molecules (called autoinducers or pheromones). These bacteria also have a receptor that can specifically detect the signaling molecule (inducer). When the inducer binds the receptor, it activates transcription of certain genes, including those for inducer synthesis. There is a low likelihood of a bacterium detecting its own secreted inducer. Thus, in order for gene transcription to be activated, the cell must encounter signaling molecules secreted by other cells in its environment. When only a few other bacteria of the same kind are in the vicinity, diffusion reduces the concentration of the inducer in the surrounding medium to almost zero, so the bacteria produce little inducer. However, as the population grows, the concentration of the inducer passes a threshold, causing more inducer to be synthesized. This forms a positive feedback loop, and the receptor becomes fully activated. Activation of the receptor induces the up-regulation of other specific genes, causing all of the cells to begin transcription at approximately the same time. This coordinated behavior of bacterial cells can be useful in a variety of situations. For instance, the bioluminescent luciferase produced by V. fischeri would not be visible if it were produced by a single cell. By using quorum sensing to limit the production of luciferase to situations when cell populations are large, V. fischeri cells are able to avoid wasting energy on the production of useless product.

LuxI : Acyl-homoserine-lactone synthase

LuxI is required for the synthesis of OHHL (N-(3-oxohexanoyl)-L-homoserine lactone) also known as VAI or N-(beta-ketocaproyl)homoserine lactone or 3-oxo-N-(tetrahydro-2-oxo-3-furanyl)-hexanamide, an autoinducer molecule which binds to luxR and thus acts in bioluminescence regulation.[8]

Catalytic activity

An acyl-[acyl-carrier-protein] + S-adenosyl-L-methionine = [acyl-carrier-protein] + S-methyl-5'-thioadenosine + an N-acyl-L-homoserine lactone.[8]

LuxR: Transcriptional activator protein luxR

The function of LuxR homologues as quorum sensors is mediated by the binding of N-acyl-L-homoserine lactone (AHL) signal molecules to the N-terminal receptor site of the proteins.[1] It is a transcriptional activator of the bioluminescence operon. It binds to the AHL autoinducer.[7]

AHL: N-acyl-homoserine lactone

AHL is a kind of signaling molecule involved in bacterial quorum sensing.[2] In Vibrio fischeri, AHL binds to the protein product of the LuxR gene and activates it. The C-terminal domain of activated LuxR relieves the repression exerted by H-NS nucleoid proteins that bind to the promoters of LuxR, LuxI and the LuxCDABEG operon, as well as to A-T-rich stretches within that operon and other genomic regions. The product of LuxI catalyses the synthesis of AHL. Thus, AHL acts as an autoinducer. Transcription of the LuxCDABEG operon results in luminescence due to the expression of LuxA and LuxB, which form a protein known as a luciferase and the expression of LuxC, D, E, and G, which are involved in the synthesis of the luciferase's substrate, tetradecanal.[11]

IPTG: Isopropyl β-D-1-thiogalactopyranoside

This compound is used as a molecular mimic of allolactose, a lactose metabolite that triggers transcription of the lac operon. Many regulatory elements of the lac operon are used in inducible recombinant protein systems.[14]

iGEM-Team XMU-China has designed a series of circuits driven by The PLlac 0-1 promoter (BBa_R0011). IPTG was used as an inducer to activate these circuits.

3.2 toxin ccdB

Toxin ccdB is a component of toxin-antitoxin (TA) module, functioning in plasmid maintenance. Cell killing by CcdB is accompanied by filamentation, defects in chromosome and plasmid segregation, defects in cell division, formation of anucleate cells, decreased DNA synthesis and plasmid loss.

LacZalpha-ccdB

The LacZ alpha-CcdB fusion protein has retained both the CcdB killer activity and the ability to alpha-complement the truncated LacZ delta M15.

ccdB vs cell DEATH

iGEM-Team XMU-China has designed and constructed a ccdB producer driven by promoter lux pR. We assumed that for circuit-regulated growth, the cell death rate is proportional to the intracellular concentration of the killer protein CcdB. For this reason, we controlled the expression level of the killer gene ccdB in three ways: (1) by using different RBSs (RBS1.0, RBS0.6, RBS0.3, RBS0.07); (2)by using different LuxR promoters(site-directed mutagenesis); (3) by using different LuxR(error-prone PCR).

3.3 RBS: Ribosome Binding Sites

A Ribosome Binding Site (RBS) is an RNA sequence found in mRNA to which ribosomes can bind and initiate translation. In order to control the expression of the killer protein ccdB, our team designed a series of bacteria population-control devices using RBSs with different strength. The cell growth and fluorescent curves corresponding to different RBSs illustrate that the bacteria population was successfully controlled at different cell densities.

3.4 Lux pR

Promoter lux pR is activated by LuxR in concert with HSL (homoserine lactone). Two molecules of LuxR protein form a complex with two molecules of the signalling compound HSL. This complex binds to a palindromic site on the promoter, increasing the rate of transcription. This promoter is used in our “killer protein producer” to regulate the expression of the killer protein ccdB. iGEM-Team XMU-China has successfully strengthened the expression of lux pR by mutating its DNA sequence. Three mutants, IR-3, IR-5, IR-3/5, were obtained by site-directed mutagenesis using 3-step PCR method.

3.5 IR-GFP

IR-GFP is a series of report devices designed for testing the performance of lux R promoters before and after mutagenesis. These four IR-GFP report devices have been transformed into E.coli string BL21 separately. These devices produced greenish tint visible by naked eyes when induced by IPTG. We measured and compared their florescent intensities at steady state. As the only difference between the four devices is Lux R promoter, the efficiency of the four Lux R promoters could be defined.

Protocols