Improvement

Our Project

This year, our project uses the light regulation system to control the synthesis of bait and toxic protein. Our photoinduced system consists of the following four parts. They are Cph8 (BBa_K592000), pcyA (BBa_I15009), ho1 (BBa_I15008) and promoter PompC (BBa_R0082).

cph8

The red light sensor (Cph8) is a fusion protein which is consisted of a phytochrome Cph1 and a histidine kinase domain, Envz-OmpR. Cph1 is the first member of the plant photoreceptor family that has been identified in bacteria. EnvZ-OmpR, a dimeric osmosensor, is a multidomain transmembrane protein. Cph1 can be inactivated under red light, Upon changes of extracellular osmolarity, EnvZ specifically phosphorylates its cognate response regulator OmpR, which, in turn, regulates the PompC. Cph8 can serve as a photoreceptor that regulates gene expression through PompC. Without red light, Cph1 is activated and it enables EnvZ-OmpR to autophosphorylate which in turn activates PompC. Under the exposure of red light, however, Cph1 is deactivated, inhibiting the autophosphorylation, thus turning off gene expression.

We insert RBS(B0035) and constitutive promoter(J23100) in front of the Cph8 sequences(K592000) to make sure that Cph8 can be expressed inside the E.coli.

pcyA+ho1

Moreover, Cph8 has to form chromophore with PCB biosynthetic genes (BBa_I15008 and BBa_I15009) in order to work, where the formation of PCB requires the gene pcyA and hol to function as accounted below. Hol is a sort of Iron red pigment oxidase which can oxidize the heme group using a ferredoxin cofactor, generating biliverdin IXalpha. And then, PcyA, a kind of ferredoxin oxidase from Synechocystis Sauv, functions to turn biliverdin IXalpha (BV) into PCB.

We connected gene pcyA（BBa_I15009）and ho1（BBa_I15008）together along with the constitutive promoter(BBa_J23100) to ensure a continuous work of the photoinduction system in our E.coli.

3. PompC

PompR is a OmpR-controlled promoter which can be positively regulated by phosphorylated OmpR. This promoter is taken from the upstream region of ompC. Phosphorylated OmpR binds to the three operator sites and activates transcription.

We insert PompR(BBa_R0082) sequences into the upstream region of our target gene int through restriction-ligation method, which together is connected to vector PSB1C3 afterwards, and therefore we are able to regulate the expression of in through the control of light.

Our Improvement Plan

According to the research, Cph8 is sensitive to red light, especially the wave length of red light is at 650nm. In order to improve the light sensitive characteristic of the Cph8 protein to satisfy the different requirements of this parts, such as regulating the efficiency of the promoter by control the wave length of the light, we plan to design an experiment to explore the sensibility of the Cph8 to wave length of 650nm nearby. Through this way we can control the output of the downstream product.

There are 20 experimental groups. We use the bandpass filter to control the wave length of the light from 550nm to 750nm. We set up every experimental group at a gradient of 20nm.

Materials

bandpass filter (550 /570…650 nm bandpass filters) , light (82 V, 300 W Philips FocusLine quartz bulb)

E. coli growth, light exposure and harvesting protocol

The protocol is performed over two consecutive days.

1. Late in the day, start a 37°C, shaking overnight culture from a −80 °C stock in a tube containing 3 mL LB medium and the appropriate antibiotics (50 μg/mL kanamycin, 50 μg/mL ampicillin and 34 μg/mL chloramphenicol).
2. After the overnight culture has grown for 10–12 h, prepare 100 mL LB medium. Add appropriate antibiotics to medium. Shake/stir the container to ensure the antibiotics are mixed well in the medium.
3. Measure the OD600 of the overnight culture.
4. Dilute the overnight culture 1mL into the 100mL LB + antibiotics. Shake/stir the container to ensure the cells are mixed well in the medium.
5. Place triangle bottles in the shaker and grow at 37°C with shaking at 250 rpm for 8 h. Use the narrow bandpass filter to set the wavelength of each bottles respectively. The intensity of light was measured in power units of watts per square meter using a EPP2000 UVN-SR calibrated spectroradiometer.
6. After 8 h of growth, harvest all test bottles by immediately transferring them into an ice-water bath. Wait 10 min for the cultures to equilibrate to the cold temperature and for gene expression to stop.
7. Approximately 1.5 h before stopping the experimental cultures, begin preparing a solution of phosphate-buffered saline (PBS; 137 mM NaCl, 2.7 mM KCl, 10 mM Na₂HPO₄, 2 mM KH₂PO₄, pH to 7.4). Prepare at least 1 mL for each culture to be measured via flow cytometry. At this time, begin preparing a 37°C water bath.
8. Filter the dissolved solution of PBS through a 0.22-μm 20-mL syringe filter.
9. Transfer 1 mL of the filtered PBS into one 5-mL cytometer tube per culture sample, and chill tubes in a rack in an ice-water bath.
10. Incubate the rack(s) of PBS + culture tubes in a 37 °C water bath for 1 h. Our measurements data is for the expression of RFP.
11. Transfer the rack(s) back into ice-water bath.
12. Wait 15 min, and then begin measuring each tube on a flow cytometer.

Expected Result

According to the existing literatures, what we expected is that the amount of the protein produced by experimental groups will increased at the early time, and then decreased later with the increasing of the light wave efficiency. The highest point is at around 650nm. According to the results, we can draw a line graph to describe the sensibility towards different wave length of Cph8. The following graph shows the tendency.

Our Findings

We constructed the PompC-RBS-RFP circuit first (see parts page), when we just transform this circuit into the E.coli Top 10, we wondrously found some of the colony became red. It indicated the colony had express RFP protein. It means without the regulation of the OmpR, the promoter PompC started the transcription of the downstream target gene. And then we did a sequencing towards the colony which expressed the RFP. The result indicated the PompC-RBS-RFP circuit led the expression of the RFP.

We detected the sensibility of red colony. We set RFP coding device（BBa_J04450）,RBS-rfp-terminator(BBa_K516032) and pSB1C3 as the control group. We plated 100uL the overnight culture on the LB medium consisting chloramphenicol (34ug/mL) and cultivated them in constant temperature foster box at 37℃. And half of them were under shading treatment. After 12 hours we observed the colony.

We found, all the plates transformed PompC-rfp and rfp became red. But the PompC-rfp only expresses RFP faintly and the differences with light or not are not obvious. It showed that the individual PompC-rfp biobrick was not sensitive to light. And it also indicated the Pompc promoter was at background level in E.coli TOP10.

Because in nature, this promoter PompC is upstream of the ompC porin gene. The regulation of ompC is determined by the EnvZ-OmpR osmosensing machinery. EnvZ phosphorylates OmpR to OmpR-P. At high osmolarity, EnvZ is more active, creating more OmpR-P. OmpR-P then binds to the low-affinity OmpR operator sites upstream of ompC.^[3]

The essence is that the EnvZ protein senses the mediun osmolarity and then forces the OmpR protein to take one of two alternative structures, which positively regulate OmpC synthesis.^[4]

So we designed an experiment to detect under the normal level of the Envz, the trend of E.coli PompC with the change of osmotic pressure.

Overnight cultures of Top10 strains transformed with PompR-rfp, rfp, pSB1C3 and RBS-rfp-Ter respectively grown at 37 °C in LB medium containing appropriated antibiotics were diluted at least 1:100 in the medium and incubated at 37 °C as fresh cultures. After their OD590 reached 0.2~0.4, the fresh culture was diluted 1 : 3 into 4 ml of LBON medium(1g Tryptone, 1g Yeast Extract in 100mL H2O). For osmolarity conditions, the cultures were diluted with NaCl supplemented medium to the final concentration of 0% ,0.25%, 0.50% and 1%(wt/vol). After 12 hours of induction, the results are as follows.

With increasing of the osmotic pressure, the expression of the rfp didn’t increase in experimental groups. So under natural condition, the expression of EnvZ-OmpR is too low to regulate the activity of PompC promoter. And then, from the pictures we can see the colony of experimental groups still became red. It shows that the existing of EnvZ-OmpR makes the PompC promoter become a little bit active under the natural conditions, the background level of the PompC is correspondingly higher. So if we want to try to control the expression of the downstream target gene of the PompC by using EnvZ-OmpR-PompC circuit, we’d better knock out the EnvZ-OmpR gene in the engineering bacteria first.