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

 
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        <h1 class="head4">Improve</h1>
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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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                     <a class="list-group-item list-group-item-action" href="#list-item-1">Motivation</a>
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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="#list-item-2">Experiments</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="#list-item-3">Wiring</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="#list-item-4">Reference</a>
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                     <a class="list-group-item list-group-item-action" href="#References">References</a>
 
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                             </br></br>
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                             <div id="Part_Improvement">
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                            <h3>Part Improvement</h3>
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                                </br></br></br></br>
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                            </br></br>
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                                <h3>Part Improvement</h3>
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                            <p class="pcontent">
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                                </br></br>
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                                During the designation of the experiment for our pH alert system, we have concluded
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                                that we should try to improve the expression of our pH-sensitive promoter, promoter
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                                gadA. The motivation for us to do this is because our team think that if we can build a
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                                system which could observe the situation in our device by looking at the color change
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                                of the medium, it should be an economic friendly as there is no more extra charges to
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                                send your sample to someway for checking and also wasting a lot of time for waiting the
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                                result. But according to the expression of promoter gadA from the previous team did, it
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                                is not enough to be observed by using our eyes.
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                                 <p class="pcontent">
 
                                 <p class="pcontent">
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                                     Based on our research, RiboJ first described as it is an insulator in genetic
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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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                                    construct but there has no any data shown this insulation effected the downstream
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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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                                    genetic parts. In 2018, there is a team have proved that RiboJ could increase the
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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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                                    expression of downstream gene and there is the team member, Kalen P Clifton#1,
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                                    Ethan M Jones#1, Sudip Paudel1, John P Marken2, Callan E Monette1, Andrew D
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                                    Halleran2, Lidia Epp1, Margaret S Saha1*. Hence, our team, NCKU_Tainan have desided
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                                    to enhance the expression and specificity of promoter gadA by adding a gene, RiboJ
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                                    at the downstream of promoter gadA.
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                                </br>
 
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                                <p class="pcontent">For the function determination, we have measured the fluorescent
 
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                                    density of the plasmid with and without RiboJ in different pH environment. We
 
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                                    incubated the bacteria in pH adjacent M9 medium and measure the fluorescent
 
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                                    intensity (absorbance: 480nm, excitation: 510 nm) in a short period of time.</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"
 +
                                            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>
  
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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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                            </br></br></br></br>
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</p></br>
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                            <h3>Experiment Result</h3>
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                                <img class="appimg col-lg-8" src="https://static.igem.org/mediawiki/2018/2/2f/T--NCKU_Tainan--home_42844661_1916518971978222_1636077502608703488_n.png">
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                                    <p class="pcontent" style="margin-top: 120px">
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                                        Figure 1: The fluorescent intensity expressed by PgadA of 24 hours in four
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                                        different pH value of M9 medium.
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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="appimg col-lg-8" src="https://static.igem.org/mediawiki/2018/4/41/T--NCKU_Tainan--home_42887276_674354272947210_4995870134384984064_n.png">
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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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                                    <p class="pcontent" style="margin-top: 120px">
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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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                                </div>
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                            </div>
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                            </br></br></br></br>
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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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                            <img class="contentimg result" src="https://static.igem.org/mediawiki/2018/9/9f/T--NCKU_Tainan--home_42912088_2204785603177136_6518878486872981504_n.png">
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                                <p class="pcontent">
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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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                                            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>                            
  
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                            <p class="pcontent">Figure 3: The fluorescent intensity between PgadA and PgadA-RiboJ in pH
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                                6 and pH 7.</p>
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                                <img class="contentimg result" src="https://static.igem.org/mediawiki/2018/f/fd/T--NCKU_Tainan--PGADA_OMPARISON.png">
  
 +
                                <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>
  
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                             </br></br></br></br>
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                             <div id="Conclusion">
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                            <h3>Conclusion</h3>
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                                </br></br></br></br>
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                            <div id="pt">
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                                <h3>Conclusion</h3>
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                                </br></br>
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                                <p class="pcontent">
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                                    </br></br>
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                                    Based on our functional determination test results, the improvement has succeeded
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                                    <p class="pcontent">
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                                    as RiboJ really could enhance the expression and specificity of downstream gene.
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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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                                    </p></br>
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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>
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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>
 +
                                    <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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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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