Difference between revisions of "Team:Tsinghua-A/Improve"

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                        Improve
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<h3>★  ALERT! </h3>
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                    <h2>Improvement of a previous part </h2>
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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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                    <p>BBa_K511401 HRH4-TEVs-GV16 Signaling Receptor MammoBlock (created by Group: iGEM11_MIT 2011-09-27)</p>
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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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                    <p>We based on this part, kept the coding sequence of HRH4 and added a new promoter ADH1, a new tag mcherry and ADH1 terminator. Then, we got our new part BBa_K2583001, in our project, its function is not related the original one, it is used to test the expression and the location of human histamine receptor H4 (HRH4) in yeast, it lays the foundation for the subsequent experiments. </p>
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<h1>Improve</h1>
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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>Gold Medal Criterion #2</h3>
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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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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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<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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                    <h3> More Information </h3>
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                    <p><a href="http://parts.igem.org/Part:BBa_K511401"><u>BBa_K511401</u></a></p>
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                    <p><a href="http://parts.igem.org/Part:BBa_K2583001"><u>BBa_K2583001</u></a></p>
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                    <h2>Experiment</h2>
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                    <h3>Fluorescence microscopy</h3>
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                    <p>After transforming the CENPK2-1C ura3::far1 his2::sst2 trp3::ste2 △ura3 Saccharomyces cerevisiae with plasmid pGADT7 contains part BBa_K2583001, 30℃ cultivate on △leu solid medium for one day. Select monoclonal and enlarge culture overnight, then observe the fluorescence under the microscope with 580nm exciting light. The red fluorescence shows the expression of HRH4-mcherry fusion protein.</p>
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                            <img src="https://static.igem.org/mediawiki/2018/a/ab/T--Tsinghua-A--improve-improveFig1.png" alt="" height="700" width="700"/>
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                            <p>Figure1. the fluorescence microscopy result of HRH4-mcherry</p>
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                    <p>In order to find out if HRH4 can be expressed correctly in yeast’s membrane, DIO, a kind of Green fluorescent probe of cell membrane, is add to the yeast. In figure 2, it shows that the most of the red HRH4-mcherry colocalized with DIO in the membrane, it indicates there are quite a few HRH4-mcherry fusion proteins have correct localization. </p>
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                            <p>Figure 2. the fluorescence microscopy result of DIO dying and HRH4-mcherry</p>
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                    <h3>Flow CytoMetry (FCM)</h3>
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                    <p>From the result of fluorescence microscopy, the expression of HRH4-mcherry fusion proteins can be observed roughly, for measuring the expression level of HRH4-mcherry, FCM is performed. From the result, a distinct peak value can be observed. Though it can’t representative all the functional HRH4 that locates in the membrane, it can be indicated that most of them are correctly located from the fluorescence microscopy result, this data is enough to support our modeling.</p>
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                    <h2>Result</h2>
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                    <p>The test of this composite part set the foundation of our project, it proved that HRH4 can be expressed in high level under the trigger of a constitutive promoter-ADH1 promoter. Besides, the HRH4 can locate in the yeasts’ membrane without adding other sequence. It can be indicated that if mcherry is removed, functional HRH4 can still be expressed and locate in the membrane.</p>
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Latest revision as of 16:48, 17 October 2018

Improve

Improvement of a previous part

BBa_K511401 HRH4-TEVs-GV16 Signaling Receptor MammoBlock (created by Group: iGEM11_MIT 2011-09-27)

We based on this part, kept the coding sequence of HRH4 and added a new promoter ADH1, a new tag mcherry and ADH1 terminator. Then, we got our new part BBa_K2583001, in our project, its function is not related the original one, it is used to test the expression and the location of human histamine receptor H4 (HRH4) in yeast, it lays the foundation for the subsequent experiments.

More Information

BBa_K511401

BBa_K2583001

Experiment

Fluorescence microscopy

After transforming the CENPK2-1C ura3::far1 his2::sst2 trp3::ste2 △ura3 Saccharomyces cerevisiae with plasmid pGADT7 contains part BBa_K2583001, 30℃ cultivate on △leu solid medium for one day. Select monoclonal and enlarge culture overnight, then observe the fluorescence under the microscope with 580nm exciting light. The red fluorescence shows the expression of HRH4-mcherry fusion protein.

Figure1. the fluorescence microscopy result of HRH4-mcherry

In order to find out if HRH4 can be expressed correctly in yeast’s membrane, DIO, a kind of Green fluorescent probe of cell membrane, is add to the yeast. In figure 2, it shows that the most of the red HRH4-mcherry colocalized with DIO in the membrane, it indicates there are quite a few HRH4-mcherry fusion proteins have correct localization.

Figure 2. the fluorescence microscopy result of DIO dying and HRH4-mcherry

Flow CytoMetry (FCM)

From the result of fluorescence microscopy, the expression of HRH4-mcherry fusion proteins can be observed roughly, for measuring the expression level of HRH4-mcherry, FCM is performed. From the result, a distinct peak value can be observed. Though it can’t representative all the functional HRH4 that locates in the membrane, it can be indicated that most of them are correctly located from the fluorescence microscopy result, this data is enough to support our modeling.

Result

The test of this composite part set the foundation of our project, it proved that HRH4 can be expressed in high level under the trigger of a constitutive promoter-ADH1 promoter. Besides, the HRH4 can locate in the yeasts’ membrane without adding other sequence. It can be indicated that if mcherry is removed, functional HRH4 can still be expressed and locate in the membrane.

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