Difference between revisions of "Team:NCKU Tainan/Improve"

 
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        <h1 class="head">Improve</h1>
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                <h1 class="head">Improve</h1>
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            </div>
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            <div class="righttitle">
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                <h6 class="subtitle">On the Shoulder of Those Who Came Before Us
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</h6>
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            </div>
 
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                     <a class="list-group-item list-group-item-action" href="#Experiment_Result">Experiment Result</a>
 
                     <a class="list-group-item list-group-item-action" href="#Experiment_Result">Experiment Result</a>
 
                     <a class="list-group-item list-group-item-action" href="#Conclusion">Conclusion</a>
 
                     <a class="list-group-item list-group-item-action" href="#Conclusion">Conclusion</a>
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                    <a class="list-group-item list-group-item-action" href="#References">References</a>
 
                     <a class="list-group-item list-group-item-action" href="#"><i class="fa fa-arrow-up fa-1x"
 
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                                 </br></br>
 
                                 </br></br>
 
                                 <p class="pcontent">
 
                                 <p class="pcontent">
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                                     After analyzing the experiment result for our pH-sensing system, we have concluded that we should try to improve one of our pH-sensitive promoters, P<sub>gadA</sub>. We found out that although P<sub>gadA</sub> can be induced under neutral and weak acidic condition. But the fluorescent intensity is too slow to be observed. We planned to increase the fluorescent intensity and shorten the time interval of induction. We thus add a RiboJ sequence at the downstream of the P<sub>gadA</sub>.  
+
                                     After analyzing the experiment result for our pH-sensing system, we have concluded that we should try to improve the pH-sensing device based on gadA promoter which is constructed by <a href="https://2016.igem.org/Team:Dundee"
 +
                                            style="color:#28ff28;">2016 iGEM Dundee team</a>, P<sub>gadA</sub> (<a href="http://parts.igem.org/Part:BBa_K1962013"
 +
                                            style="color:#28ff28;">BBa_K1962013</a>). We found out that although P<sub>gadA</sub> can be induced under neutral and weak acidic condition, the fluorescence intensity is too low to be observed. We planned to increase the fluorescence intensity and shorten the time interval of induction. Thus, we added a RiboJ sequence at the downstream of the P<sub>gadA</sub>.  
 
                                 </p></br>
 
                                 </p></br>
  
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                                     </br>
 
                                     </br>
 
                                     <p class="pcontent">
 
                                     <p class="pcontent">
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                                         Based on our research, RiboJ first described as it is an insulator in genetic construct but there has no any data shown this insulation effected the downstream genetic parts. The mechanism of RiboJ is that the upstream sequence as will as RiboJ sequence will cleave after the transcription. The cleavage of promoter and RiboJ sequence aids the design of predictable genetic constructs. In 2016, the iGEM team William and Mary has reported that RiboJ could increase the expression of the downstream gene. Hence, we decided to enhance the expression of P<sub>gadA</sub> by adding a gene, RiboJ at the downstream of promoter gadA.
+
                                         Based on our research, RiboJ was first described as an insulator in genetic construct but there has no any data shown this insulation affected the downstream genetic parts. The mechanism of RiboJ is that the upstream sequence, as well as RiboJ sequence, will cleave after the transcription. The cleavage of promoter and RiboJ sequence aids the design of predictable genetic constructs. The <a href="https://2016.igem.org/Team:William_and_Mary"
 +
                                            style="color:#28ff28;">2016 iGEM William and Mary</a> team has reported that RiboJ could increase the expression of the downstream gene. Hence, we decided to enhance the expression of P<sub>gadA</sub> by adding RiboJ at the downstream of promoter gadA.
 
                                     </p></br>
 
                                     </p></br>
 
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                                 <div id="pt">
 
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                                     </br>
 
                                     </br>
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                                     <p class="pcontent">To determine the function and compare the improvement of the parts, we have measured the fluorescent intensity (fluorescence (a.u.) / O.D.600) of the construct with and without RiboJ in different pH environment. We incubated the bacteria in pH adjusted M9 medium and measure the fluorescent intensity (absorbance: 480nm, excitation: 510 nm) in a short period of time. The pH value of M9 medium is adjusted with HCl.
+
                                     <p class="pcontent">To determine the function and compare the improvement of the parts, we have measured the fluorescence intensity (fluorescence (a.u.) / O.D.600) of the construct with and without RiboJ in different pH environment. We incubated the bacteria in pH adjusted M9 medium and measure the fluorescence intensity (absorbance: 480nm, excitation: 510 nm) in a short period of time. The pH value of M9 medium is adjusted with 1M HCl.
  
 
</p></br>
 
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                                 <img class="contentimg col-6" src="https://static.igem.org/mediawiki/2018/a/a3/T--NCKU_Tainan--wifi_pgada_flrescent.png">
 
                                 <img class="contentimg col-6" src="https://static.igem.org/mediawiki/2018/a/a3/T--NCKU_Tainan--wifi_pgada_flrescent.png">
  
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                                 <img class="contentimg col-6 fig" src="https://static.igem.org/mediawiki/2018/e/ef/T--NCKU_Tainan--wifi_GADA_NTENSITY.png">
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                                 <img class="contentimg col-6 fig" src="https://static.igem.org/mediawiki/parts/c/c6/T--NCKU_Tainan--part_gadA_RIBOJ_fluorescnet_new_.png">
  
 
                                 <p class="pcontent">
 
                                 <p class="pcontent">
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                                     Figure 1: The fluorescent intensity expressed by PgadA of 24 hours in four
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                                     Fig 1. The fluorescence intensity expressed by P<sub>gadA</sub> (<a href="http://parts.igem.org/Part:BBa_K1962013"
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                                    different pH value of M9 medium.
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                                            style="color:#28ff28;">BBa_K1962013</a>) and P<sub>gadA</sub>  with RiboJ (<a href="http://parts.igem.org/Part:BBa_K2762016"
 +
                                            style="color:#28ff28;">BBa_K2762016</a>) of 14 hours in M9 medium with four different pH value.
 
                                 </p>                               
 
                                 </p>                               
  
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                                <p class="pcontent">
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                                    Figure 2: The fluorescent intensity expressed by PgadA-RiboJ of 24 hours in
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                                    four different pH value of M9 medium.
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                                </p>
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                                 <img class="contentimg result" src="https://static.igem.org/mediawiki/2018/f/fd/T--NCKU_Tainan--PGADA_OMPARISON.png">
 
                                 <img class="contentimg result" src="https://static.igem.org/mediawiki/2018/f/fd/T--NCKU_Tainan--PGADA_OMPARISON.png">
  
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                                 <p class="pcenter">Figure 3: The fluorescent intensity between PgadA and PgadA-RiboJ
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                                 <p class="pcenter">Fig 2. The fluorescence of P<sub>gadA</sub> and P<sub>gadA</sub>-RiboJ
 
                                     in pH
 
                                     in pH
 
                                     6 and pH 7.</p>
 
                                     6 and pH 7.</p>
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                                     <p class="pcontent">
 
                                     <p class="pcontent">
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                                        Based on our functional determination test results, the improvement that we did
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                                      We observe that with the RiboJ sequence, the fluorescence intensity has risen dramatically at the 14<sup>th</sup> hour. We also compare the fluorescence at 3<sup>rd</sup>, 6<sup>th</sup>, and 9<sup>th</sup> hour. The fluorescence of the strain that contains RiboJ is more than that does not contain RiboJ. We can conclude that by adding the RiboJ sequence to the construct, the expression of fluorescence protein is increased. We can also conclude that RiboJ could also strengthen the signal and increase the specificity of the downstream gene of it. We improve the P<sub>gadA</sub> part so the difference of the surrounding pH condition can be observed easier.
−
                                        has
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                                        succeeded.
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                                        As you could see, <i>E. coli</i> that carrying promoter gadA with RiboJ (Figure
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                                        2) has
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                                        the higher fluorescent level than the <i>E. coli</i> that carrying promoter
+
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                                        gadA but without
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                                        RiboJ (Figure 1).
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                                        From our experiment result, we also could conclude that RiboJ not only can
+
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                                        enhance expression
+
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                                        but it could also strengthen the signal and increase the specificity of the
+
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                                        downstream gene of it.
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                                        So, our team has successfully improve the part of promoter gadA with the
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                                        addition of RiboJ gene.
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                             </div>
 
                             </div>
  
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                            <div id="References">
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                                <h3>References</h3>
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                                <ol>
 +
                                    <li class="smallp">Clifton, Kalen P, et al. “The Genetic Insulator RiboJ Increases Expression of Insulated Genes.” Sept. 2018, doi:10.1101/317875.</li>
 +
                                    <li class="smallp">2016 iGEM Dundee team</li>
 +
                                    <li class="smallp">2016 iGEM William and Mary</li>
 +
                                </ol>
 +
                            </div>
  
 
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Latest revision as of 00:02, 18 October 2018

Improve

On the Shoulder of Those Who Came Before Us




Part Improvement



After analyzing the experiment result for our pH-sensing system, we have concluded that we should try to improve the pH-sensing device based on gadA promoter which is constructed by 2016 iGEM Dundee team, PgadA (BBa_K1962013). We found out that although PgadA can be induced under neutral and weak acidic condition, the fluorescence intensity is too low to be observed. We planned to increase the fluorescence intensity and shorten the time interval of induction. Thus, we added a RiboJ sequence at the downstream of the PgadA.



Based on our research, RiboJ was first described as an insulator in genetic construct but there has no any data shown this insulation affected the downstream genetic parts. The mechanism of RiboJ is that the upstream sequence, as well as RiboJ sequence, will cleave after the transcription. The cleavage of promoter and RiboJ sequence aids the design of predictable genetic constructs. The 2016 iGEM William and Mary team has reported that RiboJ could increase the expression of the downstream gene. Hence, we decided to enhance the expression of PgadA by adding RiboJ at the downstream of promoter gadA.



To determine the function and compare the improvement of the parts, we have measured the fluorescence intensity (fluorescence (a.u.) / O.D.600) of the construct with and without RiboJ in different pH environment. We incubated the bacteria in pH adjusted M9 medium and measure the fluorescence intensity (absorbance: 480nm, excitation: 510 nm) in a short period of time. The pH value of M9 medium is adjusted with 1M HCl.






Experiment Result



Fig 1. The fluorescence intensity expressed by PgadA (BBa_K1962013) and PgadA with RiboJ (BBa_K2762016) of 14 hours in M9 medium with four different pH value.

Fig 2. The fluorescence of PgadA and PgadA-RiboJ in pH 6 and pH 7.





Conclusion



We observe that with the RiboJ sequence, the fluorescence intensity has risen dramatically at the 14th hour. We also compare the fluorescence at 3rd, 6th, and 9th hour. The fluorescence of the strain that contains RiboJ is more than that does not contain RiboJ. We can conclude that by adding the RiboJ sequence to the construct, the expression of fluorescence protein is increased. We can also conclude that RiboJ could also strengthen the signal and increase the specificity of the downstream gene of it. We improve the PgadA part so the difference of the surrounding pH condition can be observed easier.


References

  1. Clifton, Kalen P, et al. “The Genetic Insulator RiboJ Increases Expression of Insulated Genes.” Sept. 2018, doi:10.1101/317875.
  2. 2016 iGEM Dundee team
  3. 2016 iGEM William and Mary






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