Team:ATOMS-Turkiye/Results1
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<div class="justImage" ><img src="https://static.igem.org/mediawiki/2014/7/79/Atoms_slide_2.jpeg"</div><br/> | <div class="justImage" ><img src="https://static.igem.org/mediawiki/2014/7/79/Atoms_slide_2.jpeg"</div><br/> | ||
<div class="justImage" ><img src="https://static.igem.org/mediawiki/2014/9/90/Atoms_wb_results_2.jpg"</div><br/> | <div class="justImage" ><img src="https://static.igem.org/mediawiki/2014/9/90/Atoms_wb_results_2.jpg"</div><br/> | ||
+ | <li>His tagged Aprotinin (BPTI), SOD1, GPX1 and PLAT (tPA) was overexpressed in HEK 293T cells. 36 hours post transfection cells were collected and subjected to western blotting. Aprotinin, SOD1 and tPA were expressed. However, expression of GPX1 could not be observed. Previously, we realized that GPx-1 is a member of seleno-cystein proteins. To code seleno-cystein GPx has a internal stop codon. Expression in the absense of seleno-cystein GPX-1 expression stops at the internal stop codon thus, the his-tag at the and of GPx-1 can not be expressed. Therefore,we couldn’t see any band in westernblotting. Next , we planned to add sodiumselenite to induce production of seleno-cystein. | ||
+ | </li> | ||
<div class="justImage" ><img src="https://static.igem.org/mediawiki/2014/1/1a/Atoms_wb_results_3.jpg"</div><br/> | <div class="justImage" ><img src="https://static.igem.org/mediawiki/2014/1/1a/Atoms_wb_results_3.jpg"</div><br/> | ||
+ | <li>This is comassive blue flourorange view of blotted membrane. | ||
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+ | </li> | ||
<div class="justImage" ><img src="https://static.igem.org/mediawiki/2014/0/02/Atoms_wb_results_4.jpg"</div><br/> | <div class="justImage" ><img src="https://static.igem.org/mediawiki/2014/0/02/Atoms_wb_results_4.jpg"</div><br/> | ||
+ | <li>The expression level of beta actin is the same among samples. | ||
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+ | </li><li>This is comassive blue flourorange view of blotted membrane. | ||
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+ | </li> | ||
<div class="justImage" ><img src="https://static.igem.org/mediawiki/2014/a/a0/Atoms_wb_results_5.jpg"</div><br/> | <div class="justImage" ><img src="https://static.igem.org/mediawiki/2014/a/a0/Atoms_wb_results_5.jpg"</div><br/> | ||
+ | <li>This is comassive blue flourorange view of blotted membrane. | ||
+ | |||
+ | </li> | ||
<div class="justImage" ><img src="https://static.igem.org/mediawiki/2014/5/5d/Atoms_wb_results_6.jpg"</div><br/> | <div class="justImage" ><img src="https://static.igem.org/mediawiki/2014/5/5d/Atoms_wb_results_6.jpg"</div><br/> | ||
+ | <li>We over expressed SOD-1 and tPA with different doses of SOD-1 and tPA expression vector. SOD-1 well responded to the increasing doses. tPA gave the same response to all different doses. To induce expression of GPx-1 the cells treated with different doses of sodiumselenite and GPx-1 proportionally response to the increasing doses of sodiumselenite with single dose of expression vector. | ||
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+ | </li> | ||
<div class="justImage" ><img src="https://static.igem.org/mediawiki/2014/b/b4/Atoms_wb_results_7.jpg"</div><br/> | <div class="justImage" ><img src="https://static.igem.org/mediawiki/2014/b/b4/Atoms_wb_results_7.jpg"</div><br/> | ||
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Revision as of 01:36, 18 October 2014
Sensing
- We aimed to demonstrate its functionality by inserting it into pTRE-Luc vector.
- We expect that HRE, as an enhancer, would activate the promoter existing on the downstream region of it, depending on the level of HIF1alfa in the media which is increased in hypoxic conditions.
Expected To understand which colony our gene is inserted among the colonies that we transformated pHRE-luciferase vector, we expected the picture above when we perform PCR when we use pTRE-Luc forward and MCS reverse primers. |
Experimented From the samples we perform colony PCR by using pTRE-Luc forward and MCS reverse primers, we obtained a band in 428 bp line. This image proves that our HRE sequnce is inserted into the vector, successfully. |
Luciferase Assay
NF-kappaB (NF-kB) proteins comprise a family of structurally-related eukaryotic transcription factors that are involved in the control of a large number of normal cellular and organismal processes, such as immune and inflammatory responses, developmental processes, cellular growth, and apoptosis In some circumstances, NF-kB/IkB complex can be separated by external effects such as radiation, cellular stress, pathogens, inflammation etc. In this case, NF-kB can enter into nucleus and integrate with compatible kB-RE sites in order to initiate transcription.
KBRE partını yukarıdaki şekilde görüldüğü gibi CMVmini promotorun up stream kısmına klonladık.
NF-kappaB (NF-kB) was synthesized to GenScript™ company and it came in pUC57 plasmid.we digested it with BamHI & PstI and fosfatını antartic ALP ile uzaklaştırdık.(CIP)after that we made phenol chloroform. Then we digested our vector, pTRE-luciferase, with BamHI & PstI and again we made phenol chloroform for it. Afterwards we ligated them.(pTRE-luciferase kB-RE)
We transformated our plasmid(pTRE-luciferase kB-RE) to DH5α and made colony pcr with CMV forward and kB reverse primers. So we seperated our correct plasmid from the others which is not involved kb. Bu deney sonucunda 20-30 bp hizasında bant görmeyi hedefledik.
And we observed bands of KBRE at the correct possition of agarose gel.
Reference
As the amount of IAA needed for enhancing plant growth depends on whether our bacteria are producing the compound outside or inside the plants, we attempted to replicate these findings.
Therapy
Tissue plasminogen activator (abbreviated tPA or PLAT) is a protein involved in the breakdown of blood clots. It is a serine protease (EC 3.4.21.68) found on endothelial cells and (hücreden plazmaya salgılanır), the cells that line the blood vessels. As an enzyme, it catalyzes the conversion of plasminogen to plasmin, the major enzyme responsible for clot breakdown. Because it works on the clotting system, tPA is used in clinical medicine to treat embolic or thrombotic stroke. Use is contraindicated in hemorrhagic stroke and head trauma.
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Pellentesque habitant morbi tristique senectus et netus et malesuada fames ac turpis egestas. Vestibulum tortor quam, feugiat en ullamcorper pharetra. Vestibulum erat wisi, condimentum sed, commodo vitae, ornare sit amet, wisi. Aenean fermentum, elit eget tincidunt condimentum, eros ipsum rutrum orci, sagittis tempus lacus enim ac dui. Donec non enim in turpis pulvinar facilisis. Ut felis. Praesent dapibus, neque id cu
Pellentesque habitant morbi tristique senectus et netus et malesuada fames ac turpis egestas. Vestibulum tortor quam, feugiat en ullamcorper pharetra. Vestibulum erat wisi, condimentum sed, commodo vitae, ornare sit amet, wisi. Aenean fermentum, elit eget tincidunt condimentum, eros ipsum rutrum orci, sagittis tempus lacus enim ac dui. Donec non enim in turpis pulvinar facilisis. Ut felis. Praesent dapibus, neque id cu