Part:BBa_K559011:Experience

Characterization of BBa_K559011

In Hong-Kong_CUHK iGEM 2011 team, it is decided to use a turnable light sensor to regulate the amount of an intermediate signal, chloride ion, with the downstream chloride-sensing cassette, for fine and wide-range regulation of the target gene expression.

In order to test if our Pgad gene function properly with turnable gene expression by sensing intracellular chloride concentration, we integrated our part with BBa_K559010(Halorhodopsin complete system), which is a well characterization light-sensing ion channel for chloride ion. Also, the EGFP from biobrick BBa_E0040 was used for Pgad downstream gene expression as it is a well-characterized and easily-used part for quantitative results.

The additional biobrick BBa_K559010 was integrated with this biobrick to allow a directly proportional effect between extra- and intra-cellular chloride concentration inside cell. The light parameters used was characterized and optimized in that biobrick. The range of extracellular chloride concentration used (0-0.4 M) was characterized that there is no inhibition of cell growth.

A

B

Figure 1. The effect of sodium chloride concentration in culture medium on EGFP expression in BL21(DE3). Bacteria was excited by light with 530 nm for 210 seconds at room temperature. Excited bacteria was incubated in culture medium for additional 2 hours with 200 rpm shaking. Finally, bacteria was lyzed by sonication. EGFP produced was measured. Error bar represents SEM, n=3. The bacteria transformed with both Halorhodopsin and PgadEGFP, added NaCl and excited by 530 nm showed significant production of EGFP. A.When the amount of NaCl added increases, the production of EGFP increases. B. The western blotting image is the relative quantitative support of the evidence that GFP expression increase from 0.1 M to 0.4 M of solution NaCl addition. The control (tRNA, cell lysate) shows the consistent addition of gene amount for RTPCR, or western blotting respectively. It shows the downstream regulation gene expression by chloride ion level is possible.

The GFP expression increases from 0.1 M to 0.4 M of solution NaCl addition with all optimal parameters for our biobrick system for chloride absorption. After that, we need to characterize the light parameters (wavelength, power) on GFP expression, in order to prove our photo-tunable gene expression platform to be worked.

Figure 2. The effect of excitation wavelength on EGFP expression in BL21(DE3). Bacteria was excited by light with different wavelength for 210 seconds at room temperature. Excited bacteria was incubated in culture medium for additional 2 hours with 200 rpm shaking. Finally, bacteria was lyzed by sonication. EGFP produced was measured. Error bar represents SEM, n=3. Optimal wavelength for EGFP production is 530 nm.

Figure 3. The effect of excitation power on EGFP expression in BL21(DE3). Bacteria was excited by light with 530 nm wavelength with different power for 210 seconds at room temperature. Excited bacteria was incubated in culture medium for additional 2 hours with 200 rpm shaking. Finally, bacteria was lyzed by sonication. EGFP produced was measured. Error bar represents SEM, n=3. When excitation power increases, the production of EGFP increases.

After characterizing the parameters that can tune the gene expression level (here show the example GFP expression level), we have the model to show the relationship between gene expression amount and the intracellular chloride level.

Figure 4. The modeling curve for the relationship between gene expression amount and the intracellular chloride level. The Characterize of Pgad efficiency can be found by this curve as there is the combination of work between our biobrick halorhodopsin system BBa_K559010 with Pgad for photo-tunable gene expression. This modeling result follows classic Michaelis–Menten kinetics model. The value 895.2 is the maximum amount of GFP can be expressed in our model.

Applications of BBa_K559011

Application 1- Chloricolight turnable gene expression system

In previous light sensing concept, genetic engineers woud like to add a light sensor gene in front of the target biobrick for regulation. They reach for on/off gate or narrow range of light-regulating biobrick expression. In Hong-Kong_CUHK iGEM 2011 team, it is decided to use a turnable light sensor to regulate the amount of an intermediate signal, chloride ion, with the downstream chloride-sensing cassette, for fine and wide-range regulation of the target gene expression.

Figure 1. The illustrative comparison between the use conventional light-sensing and our turnable light sensing system on gene expression. Our method requires more steps, with chloride as an intermediate signal, but leads to advance that overcomes the problem of conventional light-sensing (photo-oxidation, fine turning of only one light parameter - intensity/ illuminated time, etc)

Advantages

• Quantitative - The quantitative data is directly from intracellular chloride level instead of extracellular signal level, so there is no need to make assumption on maximum diffusion
• Turnable Switch- Our system can show the effect that turning of light parameters (intensity, wavelength, illuminated time) can lead to turnable intracellular chloride concentration. Also, minimum light illumination, which causes no harm effect on cell, with the varying external chloride concentration provides a higher and more precise turnable expression range.
• Universal Plug-in Tool - Any biobrick/ gene that we would like to fine-control its expression level can be embeded after the chloride-sensing cassette to form a complete light turnable gene regulation system

Future Application - Computer-aided light-coupled gene expression regulation platform

In our system, we have a turnable light-sensing unit. However, we need an off-gate to deactivate gene expression once the light source is removed. When the off gate system is integrated in our biobrick, we can develop a Computer-aided light-coupled gene expression regulation platform. The computer can first control all of the input parameters (light quality and quantity, chloride ion concentration in the medium) and therefore providing a controlled gene expression level.The feedback signal (fusion GFP, etc) provides a quantitative output value of the target gene expression to the computer, and then it can fine-tune the signal.

Figure 2. Computer-aided light-coupled gene expression regulation platform. The Computer-aided light-coupled gene expression regulation platform is shown for a complete system for light-controllable gene expression system with dynamic turnable part by the feedback signal given in the gene expression. It makes a automatic light regulated quantitative gene expression platform.

References: [1]J.W. Sanders, G. Venema, and J. Kok, “A chloride-inducible gene expression cassette and its use in induced lysis of Lactococcus lactis,” Applied and environmental microbiology, vol. 63, Dec. 1997, p. 4877.

User Reviews

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Northwestern 2012 Team

We used this part to form a low-pH sensor for E. coli. That part can be found here.

We used this part in conjunction with the CLC antiporter - a Cl-/H+ channel that pumps Cl- ions into the cell at low pHs. The Pgad then detects the Cl- ions inside the cell, inducing the production of a lysis protein.

In addition, the Pgad promoter part was provided to us by the CUHK 2011 team in a non-biobrick compatible plasmid as it was unavailable from the registry. Because of this, we decided to PCR the Pgad promoter out of the incompatible plasmid and then insert the part into the pSB1C3 standard registry plasmid for other teams to use in the future. Gel electrophoresis and sequencing confirm that the length and sequence of the Pgad part.