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| | <div class="mdl-card__supporting-text" style="font-size: 125%"> | | <div class="mdl-card__supporting-text" style="font-size: 125%"> |
| | <strong>June 5</strong><br> | | <strong>June 5</strong><br> |
| − | pUAST-3xHA transform into E.coli.<br>
| + | pUAST-3xHA transform into E.coli.<br> |
| − | '''June 6''' <br> | + | <strong>June 6</strong><br> |
| | 1. Pick up the single colony, and culture it with 10 mL 2xYT medium overnight. <br> | | 1. Pick up the single colony, and culture it with 10 mL 2xYT medium overnight. <br> |
| | 2. Design sequencing primers of pUAST-3xHA.<br> | | 2. Design sequencing primers of pUAST-3xHA.<br> |
| − | '''June 7''' <br> | + | <strong>June 7</strong><br> |
| | 1. Midi extraction of plasmids pUAST-3xHA. <br> | | 1. Midi extraction of plasmids pUAST-3xHA. <br> |
| | 2. Send it to GENEWIZ to sequence.<br> | | 2. Send it to GENEWIZ to sequence.<br> |
| − | '''June 8''' <br> | + | <strong>June 8</strong><br> |
| | 1. Verify the sequencing result, it is correct.<br> | | 1. Verify the sequencing result, it is correct.<br> |
| | 2. Digest pUAST-3xHA with BamHI overnight.<br> | | 2. Digest pUAST-3xHA with BamHI overnight.<br> |
| − | '''June 9''' <br> | + | <strong>June 9</strong><br> |
| | Extraction of Drosophila genome and RNA, purify the digested vector.<br> | | Extraction of Drosophila genome and RNA, purify the digested vector.<br> |
| | + | </div> |
| | | | |
| | | | |
| − | | + | <div class="mdl-card__supporting-text" style="font-size: 150%; color:#5a5a5a"> |
| − | | + | Week2 |
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| − | | + | |
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| − | | + | |
| | </div> | | </div> |
| − | <div class="mdl-card__supporting-text" style="font-size: 115%"> | + | <div class="mdl-card__supporting-text" style="font-size: 125%"> |
| − | pUAST-3xHA transform into ''E.coli''. | + | <strong>June 12</strong><br> |
| − | </div>
| + | Reverse transcription of Drosophila RNA, then get the cDNA. |
| − | <div class="mdl-card__supporting-text" style="font-size: 125%; color:#5a5a5a">
| + | <br> |
| − | June 6 | + | <strong>June 13</strong><br> |
| − | </div>
| + | Design the primers for fragments’ PCR and fragments’ sequencing. |
| − | <div class="mdl-card__supporting-text" style="font-size: 115%">
| + | <br> |
| − | 1. Pick up the single colony, and culture it with 10 mL 2xYT medium overnight. <br>
| + | <strong>June 14</strong><br> |
| − | 2. Design sequencing primers of pUAST-3xHA.
| + | 1. Clone pleP, Gal4, Gal80ts from genome (success) and TH from cDNA (failed), purify them. <br> |
| − | | + | 2. Send them to GENEWIZ to sequence.<br> |
| − | </div>
| + | <strong>June 15</strong><br> |
| − | <div class="mdl-card__supporting-text" style="font-size: 125%; color:#5a5a5a"> | + | 1. Verify the sequencing result, it is correct. <br> |
| − | June 7
| + | 2. Ligase each of them with pLB vector and transform into E.coli. <br> |
| − | </div>
| + | 3. Clone TH from cDNA again (failed). <br> |
| − | <div class="mdl-card__supporting-text" style="font-size: 115%">
| + | <strong>June 16</strong><br> |
| − | 1. Midi extraction of plasmids pUAST-3xHA. <br>
| + | 1. Pick up the single colony, do bacteria liquid PCR. <br> |
| − | 2. Send it to GENEWIZ to sequence.
| + | 2. Send 5 positive colony to GENEWIZ to sequence. <br> |
| − | | + | 3. Design the In-Fusion primers. <br> |
| − | </div>
| + | 4. Clone TH from cDNA again (failed).<br> |
| − | <div class="mdl-card__supporting-text" style="font-size: 125%; color:#5a5a5a">
| + | </div> |
| − | June 8
| + | |
| − | </div>
| + | |
| − | <div class="mdl-card__supporting-text" style="font-size: 115%">
| + | |
| − | 1. Verify the sequencing result, it is correct.<br>
| + | |
| − | 2. Digest pUAST-3xHA with BamHI overnight.
| + | |
| − | | + | |
| − | </div>
| + | |
| − | <div class="mdl-card__supporting-text" style="font-size: 125%; color:#5a5a5a">
| + | |
| − | June 9
| + | |
| − | </div>
| + | |
| − | <div class="mdl-card__supporting-text" style="font-size: 115%">
| + | |
| − | Extraction of Drosophila genome and RNA, purify the digested vector.
| + | |
| − | </div>
| + | |
| − | | + | |
| − | | + | |
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| − | | + | |
| − | <div class="android-drawer-separator"></div>
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| − | </div>
| + | |
| − | | + | |
| − | <!-- Background -->
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| − | <div class="demo-card-wide mdl-card mdl-shadow--2dp">
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| − | <div class="mdl-card__title" style="text-align:center">
| + | |
| − | <h4 class="mdl-card__title-text" style="font-size: 250%; color:#5a5a5a">Background</h4>
| + | |
| − | </div>
| + | |
| − | <div class="mdl-card__supporting-text" style="font-size: 125%; color:#5a5a5a">
| + | |
| − | Dipteran fly damage
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| − | </div>
| + | |
| − | <div class="mdl-card__supporting-text" style="font-size: 115%">
| + | |
| − | Dipteran fly damage (stress on the animal) can be divided into direct and indirect hazards.<br><br>
| + | |
| − | Direct hazards:<br>
| + | |
| − | 1) Bite, harassment: sucking mosquitoes, sand flies, gnats, midges and flies and other insects can be stabbed vampire, was bitten at itching, severe papules-like urticaria, affecting the normal life and production of animals.<br>
| + | |
| − | 2) Cause allergic reactions: arthropod salivary glands, secretions, excretions and shedding of the epidermis have all heterologous protein which can cause allergic reactions.<br>
| + | |
| − | 3) Parasitic: parasite larvae parasite can cause myiasis, for example, after bovine fly larvae parasitic in the bovine skin, the fur is perforated because of the larvae 'growth, which will lower its use value, at the same time, myiasis can also cause the decline in the quality of beef and the sharply drop in the amount of cattle milk.<br><br>
| + | |
| − | Indirect hazards:<br> | + | |
| − | Many species of Diptera are used of vector insects in the transmission of pathogens between animals, pathogens, including bacteria, parasites, viruses and rickettsia and so on. Such as mosquito-borne malaria, filariasis, yellow fever and dengue fever, etc.<br>
| + | |
| − | Chrysopa lucidum, Chrysanthemum, Chlamydomonas, Carassius auratus, Chlamydia trachomatis, Mosquitoes, gadfly and midges and other insect’ bites can make dairy cows infected with mastitis, which is one of the common breast disease during summer and dry milk.<br><br>
| + | |
| − | Whether it is direct or indirect harm, the end result always affects the normal life and production of animals.
| + | |
| − | </div>
| + | |
| − | <div class="mdl-card__supporting-text" style="font-size: 125%; color:#5a5a5a">
| + | |
| − | Fruit fly
| + | |
| − | </div>
| + | |
| − | <div class="mdl-card__supporting-text" style="font-size: 115%">
| + | |
| − | The fruit fly Drosophila melanogaster is one of the most powerful genetically tractable model organisms. Work using Drosophila has made many valuable contributions to our understanding of animal development, behavior, and physiology and of human disease. While Drosophila is also a member of Dipteran fly.<br>
| + | |
| − | <div class="demo-card-wide mdl-card mdl-shadow--2dp imagelayout" style="position: relative">
| + | |
| − | <img src="https://static.igem.org/mediawiki/2017/9/98/2017tongji_image_description_A.jpg" alt="Drosophila life cycle" style="width:100%">
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| − | NO DESCRIPTION
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| − | </div>
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| − | </div>
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| − | <div class="mdl-card__supporting-text" style="font-size: 125%; color:#5a5a5a">
| + | |
| − | UAS-Gal4
| + | |
| − | </div>
| + | |
| − | <div class="mdl-card__supporting-text" style="font-size: 115%">
| + | |
| − | In yeast, GAL4 regulates the galactose metabolism, transcriptional activation of galactose utilization genes occurs when GAL4 binds to the upstream activation sequence (UAS) containing a varying number of a 17-mer repeat. GAL4 binds to DNA as a dimer through a Zn (2)-Cys (6) zinc finger. The N-terminal region mediates both dimerization and DNA binding and contains a nuclear localization signal, while an acidic C-terminal domain controls transcriptional activation. GAL4 directly interacts with the Tra1 component of the SAGA complex, recruiting Mediator and the general transcriptional machinery to initiate transcription. This ability to activate transcription is retained when GAL4 is expressed in other species including plants, human cell lines, zebrafish, and Drosophila.<br>
| + | |
| − | <div class="demo-card-wide mdl-card mdl-shadow--2dp" style="width:100%; position: relative">
| + | |
| − | <img src="https://static.igem.org/mediawiki/2017/d/de/2017tongji_image_description_B.png" alt="UAS-Gal4 in fruit flies" style="width:100%">
| + | |
| − | <!-- <div class="mdl-card__supporting-text" style="font-size: 115%; position: absolute; top:0px; left:0px; color:#0F0F0F">
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| − | NO DESCRIPTION
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| − | </div>
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| − | </div>
| + | |
| − | <div class="mdl-card__supporting-text" style="font-size: 125%; color:#5a5a5a">
| + | |
| − | Gal80 and Gal80ts
| + | |
| − | </div>
| + | |
| − | <div class="mdl-card__supporting-text" style="font-size: 115%">
| + | |
| − | The activity of GAL4 can be repressed by physical interaction with the yeast GAL80 protein. A dimer of GAL80 binds to the C-terminal ends of the GAL4 dimer so that, while it can still bind to a UAS sequence, it can no longer activate transcription. This interaction of GAL4 and GAL80 can be taken advantage of to refine the expression pattern of GAL4-dependent transgenes.<br>
| + | |
| − | | + | |
| − | Temporal specificity can be achieved using a temperature sensitive allele of GAL80. The Temporal and Regional Gene Expression Targeting (TARGET) system takes advantage of a variant of GAL80 that while ubiquitously expressed under control of the tubulin 1α promoter is only active at permissive temperatures.<br>
| + | |
| − | | + | |
| − | Thought to be the result of a single glycine to arginine substitution at amino acid 203 of GAL80, GAL80ts is unable to bind GAL4 at restrictive temperatures above 29 °C but retains its repressive function at the permissive temperature of 18 °C. Controlling the activity of GAL80ts through temperature shifts provides temporal control of GAL4-dependent transgene expression. Limiting expression to defined temporal windows can help to define critical periods for the effects of misexpression or rescue experiments. Temperature- sensitive alleles of GAL4 itself have been generated and tested in Drosophila, however the ease of combining tubGAL80ts with already established GAL4 drivers cause the TARGET system to be more commonly used.<br>
| + | |
| − | | + | |
| − | <div class="demo-card-wide mdl-card mdl-shadow--2dp" style="width:100%; position: relative">
| + | |
| − | <img src="https://static.igem.org/mediawiki/2017/0/08/2017tongji_image_description_C.png" alt="GAL80ts" style="width:100%">
| + | |
| − | <!-- <div class="mdl-card__supporting-text" style="font-size: 115%; position: absolute; top:0px; left:0px; color:#0F0F0F">
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| − | NO DESCRIPTION
| + | |
| − | </div> -->
| + | |
| − | </div>
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| − | <h4 class="mdl-card__title-text" style="font-size: 250%; color:#5a5a5a">References</h4>
| + | |
| − | </div>
| + | |
| − | <div class="mdl-card__supporting-text" style="font-size: 115%">
| + | |
| − | Christian Dahmann. Drosophila Methods and Protocols 2nd Edition. New York: Humana Press, 2016: 33-38.
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