Difference between revisions of "Team:HZAU-China/InterLab"

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   <div class="HZAU_content">
 
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     <div class="HZAU_item">
 
     <div class="HZAU_item">
−
       <a class="biaoti">Experiment</a>
+
       <a class="biaoti">Interlab</a>
−
       <a class="zhengwen">The function of dCas9 and gRNA</a>
+
       <a class="zhengwen">This year the HZAU-China iGEM team participated in the interlab held by iGEM official. The determination of this year
−
       <a class="zhengwen">We first construct the gRNA targeting at OriC and test if it could work as we expected. Through OD measurement we observed an inhibition of growth while dCas9 and gRNA are expressed.</a>
+
        is to obtain the transcription strength of a series parts by measuring fluorescence of these parts in DH5a E. coli.
 +
        Compared with the last year, this year we need to measure eight parts altogether, and this experiment not only provides
 +
        valuable data for the development of synthetic biology but also is a treasure experience for our lab.</a>
 +
      <a class="fubiaoti">Protocol & Results</a>
 +
       <a class="zhengwen">All experiments conducted are under the guidance of official protocol. After experiments, we filled in form and sent
 +
        back the result to measurement at iGEM dot org intime. The detail results are as following.</a>
 +
      <a class="fubiaoti">the measurement of 校准因子</a>
 +
      <a class="zhengwen">The first step of this year is to know the transformation efficiency of our plate reader, because we need to transform
 +
        our relative quantification into absolute quantification.</a>
 +
      <a class="zhengwen">The procedure is as following. Add 100 uL of LUDOX solution into 3 wells of 96-well-plate and add 100 uL of 灭菌water
 +
        into another 3 wells of 96-well-plate. Measuring the OD 600 by using Bio-Tek Synergy 2 plate reader.</a>
 +
      <a class="fubiaoti">Standard fluorescence measurement</a>
 +
      <a class="zhengwen">To make our data feasible to be compared with data from other teams, we also measured the standard curve of fluorescence.</a>
 +
      <a class="zhengwen">The procedure is as following. The supplied fluorescence powder are all suspended into 1mL PBS to final concentration
 +
        as 50uM, then series diluted into gradient concentration as 25uM, 12.5uM, 6.25uM,3.125uM until 50*2^-8 uM. By using
 +
        plate reader, a standard curve of fluorescence is obtained.</a>
 
       <img src="https://i0.hdslb.com/bfs/bangumi/71ff7c74c34eeac1c12379c40126d200233780dd.jpg_144x144.jpg" class="tu_1">
 
       <img src="https://i0.hdslb.com/bfs/bangumi/71ff7c74c34eeac1c12379c40126d200233780dd.jpg_144x144.jpg" class="tu_1">
−
       <a class="zhengwen">For further description of this synchronization method, we quantifying the number of OriC, dxs and TerC by using qPCR, the results confirms that the initiation of chromosome replication is inhibited.</a>
+
       <a class="zhengwen">after communication with other teams, we attempted to preculture luciferase at 42°C for 4 hour to get a more stabilize
 +
        result, the result is as following</a>
 
       <img src="https://i0.hdslb.com/bfs/bangumi/71ff7c74c34eeac1c12379c40126d200233780dd.jpg_144x144.jpg" class="tu_1">
 
       <img src="https://i0.hdslb.com/bfs/bangumi/71ff7c74c34eeac1c12379c40126d200233780dd.jpg_144x144.jpg" class="tu_1">
−
       <a class="zhengwen">The flow cytometric is also used to conduct the run-out experiment to quantify the synchronization efficiency.</a>
+
       <a class="zhengwen">Compared with previous results, the latter experiment shows a more reliable data so the latter one is used to the following
 +
        experiment.
 +
      </a>
 +
      <a class="fubiaoti">Cell measurement</a>
 +
      <a class="zhengwen">At last we need to measure the promoter strength of supplied 8 parts. The OD 600 and fluorescence strength is obtained
 +
        at the same time to get data of mean fluorescence per cell.</a>
 +
      <a class="zhengwen">The tested parts are as following:</a>
 +
      <ul>
 +
        <li>Positive Control (BBa_I20270)</li>
 +
        <li>Negative Control (BBa_R0040)</li>
 +
        <li>Test Device 1 (BBa_J364000)</li>
 +
        <li>Test Device 2 (BBa_J364001)</li>
 +
        <li>Test Device 3 (BBa_J364002)</li>
 +
        <li>Test Device 4 (BBa_J364003)</li>
 +
        <li>Test Device 5 (BBa_J364004)</li>
 +
        <li>Test Device 6 (BBa_J364005)</li>
 +
      </ul>
 +
      <a class="zhengwen">The experiment procedure is as following. First, the plasmid obtained from 2017 iGEM distribution is transformed into
 +
        commercial chemical competent cell of DH5a E. coli. After overnight culture, pick two single colony from the plate
 +
        into 50ml falcon tube culture containing LB with 50ug/mL 氨苄 overnight. Dilute two culture into OD= 0.01 and inject
 +
        100uL diluted culture into 96-well-plate. After measured the initiation fluorescence and OD600, the plate is cultured
 +
        in the shaker摇床 and measured the fluorescence strength and OD600 every 2 hours to get our final data.</a>
 
       <img src="https://i0.hdslb.com/bfs/bangumi/71ff7c74c34eeac1c12379c40126d200233780dd.jpg_144x144.jpg" class="tu_1">
 
       <img src="https://i0.hdslb.com/bfs/bangumi/71ff7c74c34eeac1c12379c40126d200233780dd.jpg_144x144.jpg" class="tu_1">
−
      <a class="zhengwen">According our results, we can conclude that the dCas9 and gRNA(OriC) can be used to synchronize the cell cycle of E. Coli.</a>
+
       <a class="zhengwen">The result is approximately coincide with the data online, but some high expression parts, like device 1, showed a lower growth rate than expected. We suspected that it is the overexpression that influence the growth of bacteria.</a>
−
       <a class="zhengwen">The light induced CRISPR/dCas9 at transcription level.</a>
+
       <a class="zhengwen">For more detailed data about our experiment, please feel free to download the following file.</a>
−
      <a class="zhengwen">The optimization of our CcaS-CcaR system with a higher difference in green and red light is conducted by our advisor.</a>
+
       <a class="fubiaoti">Suggestion for further interlab project</a>
−
      <img src="https://i0.hdslb.com/bfs/bangumi/71ff7c74c34eeac1c12379c40126d200233780dd.jpg_144x144.jpg" class="tu_1">
+
       <a class="zhengwen">During our experiment, we find that the recommended absorption and emission wavelength, 501~511nm, is not suitable
−
      <a class="zhengwen">The result shows the performance of this system. Next we insert the sequence of gRNA(OirC) behind the Pcpcg2, measure the OD performance under green and red light(fig).</a>
+
        for our plate reader leading to a data overflow, so we recommend to a wider range, 485~528nm, to acquire data.</a>
−
      <img src="https://i0.hdslb.com/bfs/bangumi/71ff7c74c34eeac1c12379c40126d200233780dd.jpg_144x144.jpg" class="tu_1">
+
−
      <a class="zhengwen">The results shows a difference in growth rate when in green or red light. Similarly, next we performed the qPCR and flow cytometric to further demonstrate the synchronization.</a>
+
−
      <img src="https://i0.hdslb.com/bfs/bangumi/71ff7c74c34eeac1c12379c40126d200233780dd.jpg_144x144.jpg" class="tu_1">
+
−
       <a class="zhengwen">But due to time limitation, the result is not so good to see. At last, we test if this system can make reverse regulate, here is our results.</a>
+
−
       <a class="zhengwen">The light induced CRISPR/dCas9 at protein level</a>
+
−
       <a class="zhengwen">We first constructed series of infused pMag-sdCas9 and nMag-sdCas9 with difference split methods, but results of OD measuremen do not shows any difference between them and wild type.</a>
+
−
      <img src="https://i0.hdslb.com/bfs/bangumi/71ff7c74c34eeac1c12379c40126d200233780dd.jpg_144x144.jpg" class="tu_1">
+
−
      <a class="zhengwen">Next we go back to find out where is the problem. First we test the function of pMag and nMag through Luciferase assay.</a>
+
−
      <img src="https://i0.hdslb.com/bfs/bangumi/71ff7c74c34eeac1c12379c40126d200233780dd.jpg_144x144.jpg" class="tu_1">
+
−
      <a class="zhengwen">Results shows that the function of pMag and nMag. Next we test the expression of split dCas9 to check if it is hydrolysis by SDS-PAGE.</a>
+
−
      <img src="https://i0.hdslb.com/bfs/bangumi/71ff7c74c34eeac1c12379c40126d200233780dd.jpg_144x144.jpg" class="tu_1">
+
−
      <a class="zhengwen">After inserting it into pET plasmid, the results turnout that protein is ......</a>
+
−
      <a class="zhengwen">The last reason we want to see if it is because the split method that makes the split dCas9 can’t complement. But due to some reason, we can’t finish constructing the plasmid in time and have no further results.</a>
+
−
      <a class="HZAU_supplement">Material and methods</a>
+
−
      <a class="HZAU_supplement">Plasmid construction</a>
+
−
      <a class="HZAU_supplement">The fragment was PCR amplified with pfu(company) or PrimeSTAR(Takara) according to product length. Product is recycled with gel extraction kit from qiagene after electrophoresis. Golden gate is conducted with total 10 liter containing 100 ng template, 5 liter buffer, 5U BsaI, and 10 U T4 ligase. With 50 cycles with 37 5min and 16 10min finished with 55 5min and deactivate at 98 for 5 min. Then transformed into trans5a using commercial component cell (xxx). homologous recombination to conducted using vyzyme xxxxx kit and operate following the guidebook.</a>
+
−
      <a class="HZAU_supplement">The OD measurement</a>
+
−
      <a class="HZAU_supplement">Cell are cultured overnight in LB broth containing corresponding antibiotics, and diluted into 1% fresh LB broth. When OD is reaching 0.6 add aTc of final concentration 200 ng/ ml to induce expression of dCas9 and inject it into 24 cell culture plate. Culture the plate in 37 and 150 rpm. Every hour put it into a plate reader to measure its OD.</a>
+
−
      <a class="HZAU_supplement">SDS-PAGE is conducted under the following protocol.</a>
+
−
      <a class="HZAU_supplement">The qPCR experiment is conducted using qPCR kit from takara. The sample is collected every 30min and freeze at liquid nitrogen after centrifugal. The genome is for qPCR is extracted using xxxx kit(takara) . Primer used for qPCR is as following.</a>
+
−
      <a class="HZAU_supplement">The flow cytometric is conducted with xxx. A and B are added into culture when the OD is at 0.6. After 2 hours, the riphamaycine and ceph are added to inhibit replication initiation and cell division, and cultured for another 2 hours to let the replication complete. Then the culture is stained with DAPI(roche) for 1 min and send into machine to detect the DNA concentration per cell.</a>
+
−
      <a class="HZAU_supplement">The Agarose Pad Assay</a>
+
−
      <a class="HZAU_supplement">To acquire the growth rate of bacteria for establishing the growth function, an agarose pad assay is conducted. Mix the 0.15g agarose and 10ml LB to reach the concentration of 1.5%,then heat it and add needed matter after cooling slightly.The melted agarose is supposed to be pipetted onto a cover glass(The volume is about 2.5ml.).Then put another clean cover glass on it to get a smooth flat surface after about 3 minutes. The next step is to pipette about 2.5 μL E.coli solution of 0.02 OD and get a new cover glass on it till the solution dries.It’s worth noting that maintain the favorable environment is vital.We used parafilm to avoid the evaporation and tinfoil to keep out the light when observed dCas9 4-1 which was induced by aTc. Observe the agarose pad under microscope to find the optimum bacteria, for instance, which are under the fission.The final thing you do to take and save picture 10 minutes per in 8 to 10 hours.</a>
+
 
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Revision as of 04:02, 1 November 2017

Interlab This year the HZAU-China iGEM team participated in the interlab held by iGEM official. The determination of this year is to obtain the transcription strength of a series parts by measuring fluorescence of these parts in DH5a E. coli. Compared with the last year, this year we need to measure eight parts altogether, and this experiment not only provides valuable data for the development of synthetic biology but also is a treasure experience for our lab. Protocol & Results All experiments conducted are under the guidance of official protocol. After experiments, we filled in form and sent back the result to measurement at iGEM dot org intime. The detail results are as following. the measurement of 校准因子 The first step of this year is to know the transformation efficiency of our plate reader, because we need to transform our relative quantification into absolute quantification. The procedure is as following. Add 100 uL of LUDOX solution into 3 wells of 96-well-plate and add 100 uL of 灭菌water into another 3 wells of 96-well-plate. Measuring the OD 600 by using Bio-Tek Synergy 2 plate reader. Standard fluorescence measurement To make our data feasible to be compared with data from other teams, we also measured the standard curve of fluorescence. The procedure is as following. The supplied fluorescence powder are all suspended into 1mL PBS to final concentration as 50uM, then series diluted into gradient concentration as 25uM, 12.5uM, 6.25uM,3.125uM until 50*2^-8 uM. By using plate reader, a standard curve of fluorescence is obtained. after communication with other teams, we attempted to preculture luciferase at 42°C for 4 hour to get a more stabilize result, the result is as following Compared with previous results, the latter experiment shows a more reliable data so the latter one is used to the following experiment. Cell measurement At last we need to measure the promoter strength of supplied 8 parts. The OD 600 and fluorescence strength is obtained at the same time to get data of mean fluorescence per cell. The tested parts are as following:
  • Positive Control (BBa_I20270)
  • Negative Control (BBa_R0040)
  • Test Device 1 (BBa_J364000)
  • Test Device 2 (BBa_J364001)
  • Test Device 3 (BBa_J364002)
  • Test Device 4 (BBa_J364003)
  • Test Device 5 (BBa_J364004)
  • Test Device 6 (BBa_J364005)
The experiment procedure is as following. First, the plasmid obtained from 2017 iGEM distribution is transformed into commercial chemical competent cell of DH5a E. coli. After overnight culture, pick two single colony from the plate into 50ml falcon tube culture containing LB with 50ug/mL 氨苄 overnight. Dilute two culture into OD= 0.01 and inject 100uL diluted culture into 96-well-plate. After measured the initiation fluorescence and OD600, the plate is cultured in the shaker摇床 and measured the fluorescence strength and OD600 every 2 hours to get our final data. The result is approximately coincide with the data online, but some high expression parts, like device 1, showed a lower growth rate than expected. We suspected that it is the overexpression that influence the growth of bacteria. For more detailed data about our experiment, please feel free to download the following file. Suggestion for further interlab project During our experiment, we find that the recommended absorption and emission wavelength, 501~511nm, is not suitable for our plate reader leading to a data overflow, so we recommend to a wider range, 485~528nm, to acquire data.