Difference between revisions of "Team:BNU-CHINA/Description"

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<h3>Our Project</h3>
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                <p>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）.</p>
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<br/>
  
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<h2><center>Project Description</center></h2>
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<h4>1. cph8</h4>
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<h5><center>Title:Sirens</center></h5>
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                    <p>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.</p>
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<br />
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                    <p>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 <em>E.coli</em>.</p>
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<p>Pests are seriously detrimental to crops. In order to prevent this detriment, diverse kinds of chemical pesticides were used in last decades. However, these pesticides pollute the environment and increase the pest resistance substantially. Such that, it is very important to develop environment-friendly biological pest control methods, which can specifically reduce the pest populations but do not endanger other higher animals.</p>
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<p>Our project intend to build a novel engineering bacteria, which will express organics to allure pests and synthesis specific toxalbumin to kill the pests after they devour it. Furthermore, the bacteria will initiate suicide to avoid the damage to environment. </p>
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<p>Our project concerns mainly on nematode first, regarding nematode to be the crucial pathogenic organism, which most common types are ''Meloidogyne incognita'', ''Bursaphelencus xylophilus'' and ''Cyst Nematode''.</p>
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<p>A series of terpene compounds were identified, including linalool and limonene, to be the chemo-attractants for most insects, as well as play a certain role on attracting soil nematodes. So we pick linalool and limonene as the candidates from the chemo-attractants bait pool. We intend to build new metabolic pathways synthesizing these two terpene compounds in E.coli by providing linalool synthase and limonene synthase, then validate its effects on nematodes.
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</p>  
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<br />
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<p>Two kinds of proteins are toxic for nematodes. Bace16 is the serine proteinase producing by an intracellular obligate parasites in nematodes called Bacillus sp. B16. Bace16 will inhibit the growth of nematodes by hydrolyzing their collagen and corneum. MpL is an agglutinin derived from Macrolepiota procera. Feeding nematodes with E.coli expressing MpL will prevent them attain maturity. In our project we are knocking Bace16 and rMpL in E.coli respectively. After devoured by baited nematodes, the E.coli will activate the toxalbumin expressing system to kill them specifically.
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</p>  
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<br />
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<p>After a sophisticated nematode trap system is built, we will continue and expand the development of the trap systems for other kinds of agricultural pest, aiming at building the bait and the toxalbumin database of various pests.</p>  
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</div>
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<h4>2. pcyA+ho1</h4>
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official tips
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                    <p>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.
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<p>Tell us about your project, describe what moves you and why this is something important for your team.</p>
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                    </p>
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<br />
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                    <p>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 <em>E.coli</em>.
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                    </p>
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<br/>
  
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<h5>What should this page contain?</h5>
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<h4>1.3 PompC</h4>
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<ul>
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                    <p>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.
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<li> A clear and concise description of your project.</li>
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                    </p>
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<li>A detailed explanation of why your team chose to work on this particular project.</li>
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                    <p>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.
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<li>References and sources to document your research.</li>
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                    </p>
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<li>Use illustrations and other visual resources to explain your project.</li>
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<br/>
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</ul>
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<hr/>
  
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<h3>2.  Our Improvement Plan</h3>
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                <p> 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.
 +
                </p>
 +
                <p>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.
 +
                </p>
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                <br/>
  
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<br />
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<h4>2.1 Materials</h4>
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<h4>Advice on writing your Project Description</h4>
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                    <p>bandpass filter (550 /570…650 nm bandpass filters) , light (82 V, 300 W Philips FocusLine quartz bulb)
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                    </p>
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<br/>
  
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<p>
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<h4>2.2 E. coli growth, light exposure and harvesting protocol</h4>
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We encourage you to put up a lot of information and content on your wiki, but we also encourage you to include summaries as much as possible. If you think of the sections in your project description as the sections in a publication, you should try to be consist, accurate and unambiguous in your achievements.  
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                    <figure class="text-center">
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</p>
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                        <img src="https://static.igem.org/mediawiki/2015/4/46/BNU-impro1.jpg" alt="Loss the Fig" />
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                        <figcaption>Figure 1
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                        </figcaption>
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                    </figure>
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                    <p>The protocol is performed over two consecutive days.
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                    </p>
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                    <ol>
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                        <li>
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                            <p>Late in the day, start a 37&#176;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).</p>
 +
                        </li>
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                        <li>
 +
                            <p>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.</p>
 +
                        </li>
 +
                        <li>
 +
                            <p>Measure the OD600 of the overnight culture.</p>
 +
                        </li>
 +
                        <li>
 +
                            <p>Dilute the overnight culture 1mL into the 100mL LB + antibiotics. Shake/stir the container to ensure the cells are mixed well in the medium.</p>
 +
                        </li>
 +
                        <li>
 +
                            <p>Place triangle bottles in the shaker and grow at 37&#176;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.</p>
 +
                        </li>
 +
                        <li>
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                            <p>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.</p>
 +
                        </li>
 +
                        <li>
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                            <p>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 Na<sub>2</sub>HPO<sub>4</sub>, 2 mM KH<sub>2</sub>PO<sub>4</sub>, 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&#176;C water bath.</p>
 +
                        </li>
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                        <li>
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                            <p>Filter the dissolved solution of PBS through a 0.22-μm 20-mL syringe filter.</p>
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                        </li>
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                        <li>
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                            <p>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.</p>
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                        </li>
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                        <li>
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                            <p>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.</p>
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                        </li>
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                        <li>
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                            <p>Transfer the rack(s) back into ice-water bath.</p>
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                        </li>
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                        <li>
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                            <p>Wait 15 min, and then begin measuring each tube on a flow cytometer.</p>
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                        </li>
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                    </ol>
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<br/>
  
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<p>
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<h4>2.3 expected result</h4>
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Judges like to read your wiki and know exactly what you have achieved. This is how you should think about these sections; from the point of view of the judge evaluating you at the end of the year.
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                    <figure class="text-center">
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</p>
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                        <img src="https://static.igem.org/mediawiki/2015/6/62/BNU-impro2.jpg" alt="Loss the Fig" />
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                        <figcaption>Figure 2
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                        </figcaption>
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<br />
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                    </figure>
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<h4>References</h4>
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                    <p>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.
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<p>iGEM teams are encouraged to record references you use during the course of your research. They should be posted somewhere on your wiki so that judges and other visitors can see how you though about your project and what works inspired you.</p>
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                    </p>
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<hr/>
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<h4>Inspiration</h4>
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<p>See how other teams have described and presented their projects: </p>
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<ul>
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<li><a href="https://2014.igem.org/Team:Imperial/Project"> Imperial</a></li>
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<li><a href="https://2014.igem.org/Team:UC_Davis/Project_Overview"> UC Davis</a></li>
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<li><a href="https://2014.igem.org/Team:SYSU-Software/Overview">SYSU Software</a></li>
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</ul>
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</div>
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</html>
 
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{{BNU-CHINA/article/end}}

Revision as of 18:25, 14 September 2015

Team:BNU-CHINA - 2015.igem.org

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）.


1. 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.


2. 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.


1.3 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.


2. 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.


2.1 Materials

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


2.2 E. coli growth, light exposure and harvesting protocol

Loss the Fig
Figure 1

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.


2.3 expected result

Loss the Fig
Figure 2

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.