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Characterizations of BBa_K55901

Gene and Protein

First, we need to characterize its gene size and sequence.

We use PCR with six pairs of primers and gel electrophoresis to confirm that we have properly constructed halorhodopsin complete system (BBa_K559010) (Fig. 1). All bands in each lane match their theoretical sizes. Halorhodopsin complete system then was transformed to BL21(DE). Since our halorhodopsin has been HIS-tagged, we performed western blot to confirm the expression of halorhodopsin in BL21(DE) (Fig. 2). The PCR and western blot results indicate that our biobricks is successfully constructed and halorhodopsin can be properly expressed in BL21(DE).

Figure 1. Halorhodopsin complete system (BBa_K559010) was correctly constructed. Components in the plasmid were amplified by standard PCR using corresponding primers. The PCR conditions were 95°C denature for 30s, 54 °C annealing for 15s and 72°C extension for 30s. The number of PCR cycle was 30. The PCR products of corresponding lanes are shown on the table. T7 was amplified by T7 F and T7 R, HR was amplified by HR F and HR R, 24C was amplified by 24C F and 24C R, T7 HR was amplified by T7 F and HR R, T7 24C was amplified by T7 F and 24C R and HR 24C was amplified by HR F and 24C R.

This biobrick made has to be optimized for maximize protein expression. To optimize the expression of Halorhodopsin, induction by different concentration of IPTG was carried out and SDS-PAGE was done to investigate the results. 0.10mM 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 concentration of IPTG was used to induce gene expression of Halorhodopsin when OD600 reached 0.4. After 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 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. 5). 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.

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 the bacteria 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 additional, we also proved that function of halorhodopsin is light depended (Fig. 7).

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 that light, IPTG and NaCl are all required for the large amount 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.

We followed MQAE method to determine intracellular chloride ion concentrations after the cells were treated with different wavelength, intensity and duration of illumination. Previous study shows that halorhodopsin mainly absorbs photons with wavelength around 600 nm^-1. Our data agree with this conclusion and we find that halorhodopsin functions with maximum efficiency at 530 nm (Fig. 6) and changing the wavelength can significantly reduce its efficiency.

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 wavelength for 2 minutes under confocal microscope. 200 μl of excited bacteria was collected and disrupted. Its intracellular chloride ion concentration was measured by MQAE. Error bar represents SEM.

Then we fixed the wavelength to 530 nm and measured the pumping efficiency of halorhodopsin under different light intensity. First we tested the survival of halorhodopsin-transformed E. coli under different light intensities (Fig. 3). The light intensity was indicated by the percentage of full LASER power 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 intensity (Fig. 4). 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. 5). 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 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.

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.

Applications of BBa_K559010

Application 1 - Computer-aided light-coupled gene expression regulation platform

We apply this biobrick system for the Computer-aided light-coupled gene expression regulation platform.

Figure 1: 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.

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.

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.

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