Part:BBa_K559010:Experience

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Protein

First, we need to characterize the protein expression of our biobrick.

This biobrick has to be optimized for maximize protein expression. To optimize the expression of Halorhodopsin, induction by different concentrations of IPTG was carried out and SDS-PAGE was done to investigate the results. 0.1mM concentration of IPTG was found to be optimum to induce Halorhodopsin expression. We thus decided to use this condition to induce expression afterwards.

Figure 2. Optimization of Halorhodopsin expression in BL21(DE). BL21(DE) was transformed according to standard protocol. Bacterial culture grew from a single colony at 37℃ in the presence of chloramphenicol. Different concentrations of IPTG were used to induce gene expression of Halorhodopsin when OD600 reached 0.4. After carrying out IPTG induction for 4 hours, bacterial samples were collected and disrupted. Bacterial samples were analyzed by 12% SDS-PAGE. Halorhodopsin was HIS-tagged and was detected by anti-His antibody with dilution of 1:5,000. Secondary antibody was anti-mouse antibody with dilution of 1:2,000 was used. Bacterial proteins in lysate stained by coomassive brilliant blue served as loading control.

Chloride ions absorption

In our application, we would like to make use of this biobrick system to absorb chloride ions in various concentrations of salt solution. In order to determine whether halorhodopsin would affect the survival of E. coli, we performed an assay to examine the growth curve of BL21(DE) transformed with the complete halorhodopsin system (BBa_K559010) in different sodium chloride concentrations (Fig. 3). Our data show that the bacteria expressing halorhodopsin grew normally in LB medium with additional sodium chloride concentration ranged from 0 M to 0.4 M. When the sodium chloride concentration was above 0.4 M, the growth of the bacteria was inhibited due to extremely high salinity conditions. Halorhodopsin does not affect the growth and survival of E. coli.

A.

B.

Figure 3. Growth curve of bacteria under different NaCl concentration. A. BL21(DE) without transformation were grew from a single colony at 37°C. 0.1 mM of IPTG was added. B. BL21(DE) was transfomrmed according to standard protocol. Bacterial culture grew from a single colony at 37°C. 0.1 mM of IPTG was used to induce Halorhodopsin expression. 1% (v/v) inoculum was used as start culture at 0 hour. The change in OD600 was monitored. At 0-4 hour time point (enough for our project of Hong-Kong_CUHK iGEM2011), there are overlapped curves from NaCl conc. 0- 0.4M (green area), showing there is no effect on cell growth. For NaCl conc. above 0.5M (red), there is inhibition of cell growth. Accurate determination of NaCl conc. having no effect on cell growth between NaCl conc. of 0.4-0.5M has to be done. Up to now, Hong-Kong_CUHK iGEM2011 team decide to use 0.4M external NaCl solution in optimization of chloride absorption.

Figure 4. Halorhodopsin expressing bacteria absorbed chloride ions. BL21(DE) was transformed and bacterial culture grew from a single colony at 37°C in the presence of light. 0.1 mM of IPTG and 0.4 M of NaCl were included in the culture medium. Intracellular Cl- was determined by MQAE after 4 hours induction.

Figure 5. Halorhodopsin expressing bacteria absorbed chloride ions under different condition. BL21(DE) was transformed and bacterial culture grew from a single colony at 37°C under different conditions. Bacterial samples were collected when OD600 reached 0.4. Bacteria from different growth conditions were disrupted. The intracellular Cl- was determined by MQAE. Light: bacteria grew in the presence of light. IPTG: 0.1 mM of IPTG was used to induce gene expression. NaCl: the LB contained 0.4 M of NaCl. “+” indicates the corresponding component was included. “-” indicates the corresponding component was missed.

From the results of Figure 5, three parameters, namely light, IPTG and NaCl, are all required for the large amount of increase in the chloride ion absorption by halorhodopsin.The comparative increase in set up 4, 5 and 7 are due to the addition of NaCl, which causes a greater diffusion of chloride ions into the cell.

Optimization

To test the function of halorhodopsin, we performed MQAE assay to measure intracellular chloride concentration right after light illumination to bacterial samples (Fig. 6, Fig. 7). After light illumination, the bacteria expressing halorhodopsin have significantly higher intracellular chloride concentrations compared with those without halorhodopsin. Our data show that halorhodopsin pumps chloride ions from medium into bacteria during light illumination. In conclusion, our biobricks can function properly in E. coli. In addition, we also proved that function of halorhodopsin is light dependent (Fig. 6-9).

First, we need to find which spectrum of wavelength lead to the best chloride ion absorption. We use monochromatic LASWER light as the light source. We chose the representative purple (405nm), green (530nm) and red (670nm).

Figure 6. Halorhodopsin expressing bacteria absorbed chloride ions induced by laser with different wavelengths. BL12(DE) was transformed by BBa_K559010 and the bacterial culture grew from a single colony in LB with 0.4 M of NaCl at 37°C in the absence of light. Bacterial samples were collected when OD600 reached 0.4. 500 μl of bacterial sample was excited by 20% laser power with different wavelengths for 2 minutes under confocal microscope. 200 μl of excited bacteria was collected and disrupted. Its intracellular chloride ion concentration was measured by MQAE. Halorhodopsin functioning for chloride absorption has the maximum efficiency at 530 nm. Error bar represents SEM.

Therefore, we fixed the wavelength to 530 nm and measured the pumping efficiency of halorhodopsin under different light intensities.

We tested the survival of halorhodopsin-transformed E. coli under different light intensities (Fig. 7). The light intensity was indicated by the percentage of full LASER power (25mW) supply under confocal microscope. The growth of transformed E. coli was not significantly affected when they were exposed to the light intensity below 25 percent of full power. E. coli could still grow normally when they were exposed under the light with intensity between 30 percent and 40 percent of the full power. However, when the LASER power exceeded 40 percent, the bacterial growth was significantly inhibited. In the next step, we examined the chloride absorption efficiency of halorhodopsin under different light intensities (Fig. 7).

Figure 7. Bacterial growth after exposure to different power of laser with wavelength 530 nm. Transformed BL21(DE) grew in LB with 0.4 M of NaCl. 500 μl of bacteria culture was used for laser exposure when OD600 reach 0.4. Bacteria were exposed to different laser power for 2 minutes. After the exposure, 200 μl of the bacteria was cultured in 2 ml of LB. OD600 was determined immediately after inoculation (0 hour) and after 2 hour grew in LB. Table shows the value of OD600 at corresponding time point. Data was expressed as mean ± SEM.

Second, we found that the highest chloride pumping efficiency appeared at 25 percent of full power. When the intensity kept increasing, the intracellular concentration dropped due to death of E. coli. Furthermore, we studied the relation between intracellular chloride ion concentration and illumination duration under 25 percent of full power (Fig. 8). The highest intracellular chloride concentration was 0.55 M after samples was illuminated for 210 seconds. Afterwards intracellular chloride ions kept decreasing due to cell death caused by high LASER power.

Figure 8. Laser (530 nm) induced Chloride ion absorption. BL12(DE) was transformed and a single colony was picked to grow in LB with 0.4 M of NaCl. 500 μl of bacterial sample with OD600 reached 0.4 was excited by laser (530 nm) with different LASER power (%). 200 μl of excited bacteria was collected and disrupted. The intracellular concentration of chloride ion was determined by MQAE.

Figure 9. Chloride ions absorption responded differently with the length of laser (530 nm) exposure. BL12(DE) was transformed and a single colony was picked to grow in LB with 0.4 M of NaCl. 500 μl of bacterial sample with OD600 reached 0.4 was excited by LASER (530 nm) with different exposure time. The LASER power was fixed at 25%. 200 μl of excited bacteria was collected and disrupted. The intracellular concentration of chloride ion was determined by MQAE.

In conclusion, our data present the feasibility where intracellular chloride concentration can be accurately controlled by the wavelength, intensity and duration of light illumination for this biobrick. And this made the unique feature, that is a light-turnable chloride absorption, leading to some downstream applications listed below.

Applications of BBa_K559010

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, to 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)

Figure 2. The result shows that Pgad chloride sensing cassette can be induced by different concentration of sodium chloride addition, with the controlled level of GFP expression, with the relative quantity shown in the western blot image. The control (tRNA, cell lysate) shows the consistent addition of gene amount for RTPCR, or western blotting respectively. The GFP expression increase from 0.1 M to 0.4 M of solution NaCl addition with all optimal parameter for our biobrick system for chloride absorption. It shows the downstream regulation gene expression by chloride ion level is possible.

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 Amplifier - 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 that cause no harm effect on cell, with the varying external chloride concentration provides a higher and more precies 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

Application 2- Mixing-entropy battery Our biobrick in the bacteria allows capturing of solar energy to generate potential gradient of chloride ion in the solution, this in turn can convert into electricity though two special equipments.

1. Mixing-entropy electrodes 2.Power management system

The first one is a specialized electrode that can sense the entropy change in the solution The second one can stablize electricity and boost the small voltage up to a applicable voltage, for switching on LEDs in our examples.

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

In our system, we have a turnable light-sensing unit. However, we need a off-gate to deactivate gene expression once 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 the input parameter (light quality and quantity, chloride ion concentration in medium), and lead to controlled gene expression level. The feedback signal (fusion GFP, etc) provide a quantitative output value of the target gene expression to the computer, then it can fine-tune the signal.

Figure 3: 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

We may be able to use this system for Solar Electricity Generation. The bacteria with the halorhodopsin gene in this biobrick can take up light energy for chloride absorption, leading to increase in intracellular chloride ion concentration inside the bacteria. The bacteria will be translocated into low salt environment, leading to release of chloride ion by diffusion. The potential different in the above two step generates voltage and current.

The design can generate potential change as stated in theory. More experimental data is needed for the function efficiency, output voltage and current, etc.

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