Team:XMU-China/Project

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Contents 1 Project Name 2 Project Description 3 Approach 4 3 Background 5 3.1 Quorum Sensing 6 3.2 toxin ccdB 7 3.3 RBS: Ribosome Binding Sites 8 3.4 Lux pR 9 3.5 IR-GFP 10 Protocols

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

1.Isolation of Plasmid

Using the Procedure for GenEluteTM Plasmid Miniprep Kit

1. Collect 1-5 mL bacterium fluid in 1.5 mL centrifuge tube，and centrifuge fluid at 12000 r/min

1. Resuspend cells. Discard the supernatant and completely resuspend the bacterial pellet with 250 µl of the Resuspension Solution

1. Lyse cells. Lyse the resuspended cells by adding 250 µl of the Lysis Solution

1. Neutralize. Precipitate the cell debris by adding 350 µl of the Neutralization/Binding Solution, and centrifuge fluid at 12000r/min

1. Load cleared lysate. Transfer the supernatant from step 4 to the spin column. Centrifuge at 12000r/min for 1 minute, and then discard the filtrate

1. Optional wash. Add 500 µl of the Optional Wash Solution to the column. Centrifuge at 12000 r/min for 1 minute. Discard the filtrate

1. Wash column. Add 500 µl of the diluted Wash Solution to the column. Centrifuge at 12000r/min for 1 minute.

1. Elute DNA. Transfer the column to a new collection tube. Add 50~100 µl of Eluent Solution to the column. Centrifuge

at 12000 r/min for 1 minute. The DNA is now present in the filtrate and is ready for immediate use or storage at -20℃

2.Reaction system of restriction endonuclease

Figure

System1、2、3 and 4 are used for Standard BioBrick Assembly .System 5 and 6 are used for Restriction analysis. Digestion of sample: at least 500 ng DNA / 10 µL volume. Digest for 4 h at 37 °C, afterwards inactivated by adding 10x loading buffer and standing for 10 min at room temperature.

3. Standard BioBrick Assembly

1. Digestion of insert: 2 μg~5 μg DNA / 100 µL volume, 10x H buffer, EcoRI, SpeI. Digestion and inactivation. Clean up the insert via gel electrophoresis. When cutting the insert out of the gel, try to avoid staining or exposure to ultraviolet light of the insert.

1. Digestion of vector: 2 μg~5 μg DNA / 100 µL volume, 10 x M buffer, EcoRI, XbaI. Digestion and inactivation. Clean up the insert via gel electrophoresis. When cutting the insert out of the gel, try to avoid staining or exposure to ultraviolet light of the insert.

4. Suffix Insertion

1. Digestion of insert: 2 μg~5 μg DNA / 100 µL volume, 10x M buffer, XbaI, PstI. Digestion and inactivation. Clean up the insert.

1. Digestion of vector : 2 μg~5 μg DNA / 100 µL volume, 10x H buffer, SpeI, PstI. Digestion and inactivation. Clean up the vector.

5. Ligation

1. After digestion and clean-up, the next step is ligation. Overnight ligation at 16°C. Table 2 is the system of ligation.

6. Transformation

1. Preparation of competent E.coli cells

1. Add 10 µL plasmid to 100 µl competent cells in centrifuge tube

1. Store tube on ice for 20-30 minutes

1. Water bath for 90s at 42℃

1. Put the tube on ice for 1-2min

1. Add 790 µL LB，and cultivation for 1h at 37 ℃，then plate on selective LB-Medium.

7. Restriction analysis

1. Pick one colony with a sterile tip and cultivation in 20ml LB for overnight at 37 ℃

1. Isolation of Plasmid

1. Digest BioBrick，the system of Restriction analysis refer to table1

1. Gel electrophoresis：add 2 µL loading buffer to digestion mixture. An agarose concentration is 1 %.

8. PCR -RBS1.0-luxR-TT-lux pR-

Design Primers using the primer 5

Primer：

2621-F：5'-GCTCTAGAGAAAGAGGAGAAATACT-3'

2621-R：5'-AAAACTGCAGCGGCCGCTACTA-3'

9. Cell growth

1. 100µL suspension was inoculated from a Glycerin tube into 20ml fresh LB and incubated overnight at 37℃ and 250 r.p.m.

1. 100µL suspension was inoculated again from step1 into 50ml fresh LB and incubated at 37℃ and 250r.p.m

1. IPTG was added when A600≈0.6-0.8

1. 1 ml suspension was taken on every sample taken time. 3 samples were taken in each time.

1. Diluted each sample to 10-6(Sometimes 10-5),and then plate on selective LB-Medium.

1. After 12h, count the number of CFU on the plate on different time point and then draw the cell growth curve.

10. Determining fluorescence intensity

1. Add IPTG when A600 0.6~0.8.

1. Cool the culture 10 minutes on ice.

1. Centrifuge at 6000rpm. Wash it with pre-cooled PBS buffer.

1. Use fluorescence spectrophotometer tomeasure the fluorescenceof GFP:

1. Before measuring, dilute the bacteria with PBS buffer so that it can be within the measuring #range. Set excitation wavelength 491 nm, emission wavelength 511 nm.

1. Transfer the measured bacteria in a new centrifuge tube and measure the OD of the bacteria.

11. Site Directed Mutagenesis

Figure

figure

12. Error prone PCR

figure

figure