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

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                <h1 class="head">Improve</h1>
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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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                <div id="sidelist" class="list-group">
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                    <a class="list-group-item list-group-item-action" href="#Part_Improvement">Part Improvement</a>
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                    <a class="list-group-item list-group-item-action" href="#Experiment_Result">Experiment Result</a>
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                    <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>
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                    <a class="list-group-item list-group-item-action" href="#"><i class="fa fa-arrow-up fa-1x"
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                            <div id="Part_Improvement">
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                                </br></br></br></br>
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                                <h3>Part Improvement</h3>
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                                </br></br>
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                                <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 the pH-sensing device based on gadA promoter which is constructed by <a href="https://2016.igem.org/Team:Dundee"
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                                            style="color:#28ff28;">2016 iGEM Dundee team</a>, P<sub>gadA</sub> (<a href="http://parts.igem.org/Part:BBa_K1962013"
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                                            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>.
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                                </p></br>
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                                <div id="pt">
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                                    </br>
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                                    <p class="pcontent">
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                                        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"
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                                            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.
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                                    </p></br>
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                                <div id="pt">
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                                    </br>
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                                    <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.
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                            <div id="Experiment_Result">
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                                </br></br></br></br>
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                                <h3>Experiment Result</h3>
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                                </br></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">
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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">
  
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                                <p class="pcontent">
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<h3>★  ALERT! </h3>
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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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<p>This page is used by the judges to evaluate your team for the <a href="https://2018.igem.org/Judging/Medals">medal criterion</a> or <a href="https://2018.igem.org/Judging/Awards"> award listed below</a>. </p>
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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"
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<p> Delete this box in order to be evaluated for this medal criterion and/or award. See more information at <a href="https://2018.igem.org/Judging/Pages_for_Awards"> Instructions for Pages for awards</a>.</p>
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                                            style="color:#28ff28;">BBa_K2762016</a>) of 14 hours in M9 medium with four different pH value.
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                                <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">Fig 2. The fluorescence of P<sub>gadA</sub> and P<sub>gadA</sub>-RiboJ
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                                    in pH
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                                    6 and pH 7.</p>
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                            <div id="Conclusion">
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<h1>Improve</h1>
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                                </br></br></br></br>
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<p>For teams seeking to improve upon a previous part or project, you should document all of your work on this page. Please remember to include all part measurement and characterization data on the part page on the Registry. Please include a link to your improved part on this page.</p>
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                                <h3>Conclusion</h3>
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                                <div id="pt">
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                                    </br></br>
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                                    <p class="pcontent">
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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.
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                                    </p></br>
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                                </div>
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                            </div>
  
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<h3>Gold Medal Criterion #2</h3>
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                            <div id="References">
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<p><b>Standard Tracks:</b> Create a new part that has a functional improvement upon an existing BioBrick part. The sequences of the new and existing parts must be different. You must perform experiments with both parts to demonstrate this improvement. Document the experimental characterization on the Part's Main Page on the Registry for both the existing and new parts. Both the new and existing Main Page of each Part’s Registry entry must reference each other. Submit a sample of the new part to the Registry.
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                                <h3>References</h3>
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                                <ol>
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                                    <li class="smallp">Clifton, Kalen P, et al. “The Genetic Insulator RiboJ Increases Expression of Insulated Genes.” Sept. 2018, doi:10.1101/317875.</li>
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                                    <li class="smallp">2016 iGEM Dundee team</li>
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                                    <li class="smallp">2016 iGEM William and Mary</li>
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                                </ol>
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                            </div>
  
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The existing part must NOT be from your 2018 part number range and must be different from the part documented in bronze #4.
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                            </br></br></br></br></br></br>
  
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<br><br>
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                        </div>
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<b>Special Tracks:</b> Improve the function of an existing iGEM project (that your current team did not originally create) and display your achievement on your wiki.</p>
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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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