Difference between revisions of "Team:SCUT-China/Protocols"

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               <img class="img" src="https://static.igem.org/mediawiki/parts/b/b7/SCUT2015_China_PCR1.jpg."  />  
 
               <img class="img" src="https://static.igem.org/mediawiki/parts/b/b7/SCUT2015_China_PCR1.jpg."  />  
 
               <p>3.Heat mixture to 65°C for 5 min and quick chill on ice. Collect the contents of the tube by brief centrifugation and add:</p>
 
               <p>3.Heat mixture to 65°C for 5 min and quick chill on ice. Collect the contents of the tube by brief centrifugation and add:</p>
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              <img class="img" src="https://static.igem.org/mediawiki/parts/b/b7/SCUT2015_China_PCR2.jpg."  />
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            <p>Ischemic and hypertensive heart disease were the leading causes of death in the world over the past decade. Over 7.4 million people were dead of these diseases in 2012 and the number is increasing. Patients and their families suffer from the diseases. It also left a heavy burden to government and society. This summer, we tried to solve the problem with our knowledge in synthetic biology.</p>
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              <p>4.Mix contents of the tube gently and incubate at 37°C for 2 min.</p>
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            <p>The discovery of Nitric oxide (NO) as an endogenous signaling molecule and as a mediator of the cardiovascular effects was acknowledged by the Nobel Prize in Physiology/Medicine in 1998. NO activates sGC by binding to its prosthetic heme group and thereby catalyzing the synthesis of cGMP. As a critical second messenger molecule, cGMP regulates many vital homeostatic mechanisms, i.e. endothelial cell permeability, vascular smooth muscle contractility and cardiomyocyte hypertrophy. </p>
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              <p>5.Add 1 µl (200 units) of M-MLV RT,and mix by pipetting gently up and down.</p>
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              <p>6.Incubate 50 min at 37°C.</p>
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            <p>Understanding of the NO-sGC-cGMP pathway has initiated significant translational interest, but this was almost exclusively embodied by the use of NO donors and PDE5 inhibitors. NO donors are not ideal for chronic treatment of heart diseases because of partial patient response, development of tolerance over time and short retention in the plasma. The use of PDE inhibitors is also likely to be limited as the efficacy of such molecules is dependent on endogenous cGMP generation. We therefore thought that it may be beneficial to ‘think outside the box’ to target the NO-sGC-cGMP axis using molecular medicines.</p>
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              <p>7.Inactivate the reaction by heating at 70°C for 15 min.</p>
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Revision as of 16:10, 18 September 2015

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Description

1. Cells transfectiob

1. Seed cells to be 40% confluent at a 35mm culture dish.

2. Dilute 10ul lentiviral vector in 1ml DMEM medium containing 10% FBS

3. Withdraw culture medium from 35mm culture dish.

4. Add vector-DMEM complex to cells

5. Incubate for 15 hours.

6. Withdraw vector-DMEM complex from culture dish.

7. Add 2ml DMEM medium containing 10% FBS to cells and incubate for 10 hours

8. Observe the cells under Inverted fluorescence microscope.

2. RT-PCR

RNA extraction

1.Add trizol (3ml per culture dish);

2.Keep portions in centrifuge tube(1ml per centrifuge tube)

3.Homogenized by pipetting several times.

4.Incubate samples for 5 min at room temp.

5.Add chloroform (1/5 volume of trizol; e.g. 0.2ml to 1ml)

6.Shake for 15sec.

7.Incubate samples for 5 min at room temp.

8.Centrifuge11.5G, 15 min, 4 ℃.

9.Transfer 0.5ml aqueous phase to a new centrifuge tube.

10.Add isopropanol (1/2 volume of trizol; e.g. 0.5ml to 1ml)

11.Reverse blending.

12.Incubate samples for 10 min at room temp.

13.Centrifuge11.5G, 10 min, 4 ℃.

14.Discard the supernatant.

15.Add 70% EtOH (1 volume of trizol; e.g. 1ml to 1ml ,add & vortex briefly)

16.Centrifuge11.5G, 5 min, 4 ℃.

17.Discard the supernatant.

18.Air-dry pellet for 2-5min.

19.Add 20μlRNase free water and store in -70℃ environment.

20.Determine RNA content by UV spectrophotometry.

21.Electrophoresis of RNA.

Two-Step RT-PCR STEP1:Reverse Transcription

1.Assemble the reaction on ice. Add the enzyme last.

2.Add the following components to a nuclease-free microcentrifuge tube.

3.Heat mixture to 65°C for 5 min and quick chill on ice. Collect the contents of the tube by brief centrifugation and add:

4.Mix contents of the tube gently and incubate at 37°C for 2 min.

5.Add 1 µl (200 units) of M-MLV RT,and mix by pipetting gently up and down.

6.Incubate 50 min at 37°C.

7.Inactivate the reaction by heating at 70°C for 15 min.

3. Project

3.1 Over Expression of sGC

Dysfunction of NO signaling is related to many pathological disorders, such as myocardial hypertrophy, arterial hypertension, pulmonary hypertension, heart failure, atherosclerosis and restenosis. Soluble guanylate cyclase (sGC) is a critical enzyme in NO-sGC-cGMP pathway, which catalyzes the production of cGMP from GTP. In this project, we aimed to up-regulate the concentration of cGMP by the overexpression of sGC to enhance the function of NO signaling pathway. SGC is a heterodimeric protein, contains two subunits: α and β. Both α and β subunits have a few different isoforms. We overexpressed the α3 (GUCY1A3) and β3 (GUCY1B3) isoforms because they are the most abundant forms in the cardiovascular system.

Crystal structure of the heterodimeric catalytic domain of wild-type human soluble guanylate cyclase.

(Sources:http://www.rcsb.org/pdb/pv/pv.do?pdbid=4NI2&bionumber=1# )

3.2 Silence the PDE5A

cGMP is catabolized to 5’GMP by specific members of the phosphodiesterase (PDE) superfamily. Our goal to increase cGMP synthesis by overexpressing sGC was achieved, but this may also lead to increased cGMP catabolism through the up-regulation of PDE5A. Thus, we created novel circuit consisting U6 promoter followed by PDE5A shRNA to enhance the effects of sGC overexpression.

3.3 On-Off: Hypoxia-Inducible Promoter

Absolutely,the level of cGMP will be up regulated when we enhance cGMP synthesis and block its degradation at the same time.If the devices “overexpress sGC” and “silence PDE5A” work in myocardium cells overtime,it may lead several bad effects we don’t expect.To achieve controllable up-regulation of cGMP level in the cell, we also need a switch to up regulate cGMP only in ischemia or hypoxia situation.

We found that in mammalian cells, there is already a hypoxia-inducible system. The hypoxia-inducible factor (HIF) activates transcription via binding to highly variable hypoxia-responsive elements (HREs) that are composite regulatory elements comprising the conserved HIF-binding site (HBS) with an A/GCGTG core sequence and a highly variable flanking sequence. Space between HBSs, distance from the core promoter, and orientation of HBSs are all connected with hypoxia-responsive activity. Following these rules, we design a HRE sequence and inserted it into the CMV promoter, ahead of the TATA box. We name the reconstructed CMV promoter as hypoxia-inducible promoter. To examine its function, we test an original CMV promoter submitted by the team Freiburg in 2012 as negative control,BBa_k747096.Thus, we improved the characterization of a previously existing basic part.

4. Referrence

1. Gheorghiade M, Marti CN, Sabbah HN, Roessig L, Greene SJ, Bohm M, Burnett JC, Campia U, Cleland JG, Collins SP et al: Soluble guanylate cyclase: a potential therapeutic target for heart failure. Heart failure reviews 2013, 18(2):123-134.

2. Sharina IG, Cote GJ, Martin E, Doursout MF, Murad F: RNA splicing in regulation of nitric oxide receptor soluble guanylyl cyclase. Nitric oxide : biology and chemistry / official journal of the Nitric Oxide Society 2011, 25(3):265-274.

3. Marinko M, Novakovic A, Nenezic D, Stojanovic I, Milojevic P, Jovic M, Ugresic N, Kanjuh V, Yang Q, He GW: Nicorandil directly and cyclic GMP-dependently opens K channels in human bypass grafts. Journal of pharmacological sciences 2015.

4. Ikoma E, Tsunematsu T, Nakazawa I, Shiwa T, Hibi K, Ebina T, Mochida Y, Toya Y, Hori H, Uchino K et al: Polymorphism of the type 6 adenylyl cyclase gene and cardiac hypertrophy. J Cardiovasc Pharmacol 2003, 42:S27-S32.

5. Hodges GJ, Gros R, Hegele RA, Van Uum S, Shoemaker JK, Feldman RD: Increased blood pressure and hyperdynamic cardiovascular responses in carriers of a common hyperfunctional variant of adenylyl cyclase 6. The Journal of pharmacology and experimental therapeutics 2010, 335(2):451-457.

6. Vandamme J, Castermans D, Thevelein JM: Molecular mechanisms of feedback inhibition of protein kinase A on intracellular cAMP accumulation. Cellular signalling 2012, 24(8):1610-1618.

7. Gros R, Van Uum S, Hutchinson-Jaffe A, Ding Q, Pickering JG, Hegele RA, Feldman RD: Increased enzyme activity and beta-adrenergic mediated vasodilation in subjects expressing a single-nucleotide variant of human adenylyl cyclase 6. Arteriosclerosis, thrombosis, and vascular biology 2007, 27(12):2657-2663.

8. Roth DM: Adenylyl Cyclase Increases Survival in Cardiomyopathy. Circulation 2002, 105(16):1989-1994.

9. Kuhn M: Structure, regulation, and function of mammalian membrane guanylyl cyclase receptors, with a focus on guanylyl cyclase-A. Circulation Research 2003, 93(8):700-709.

10. Takimoto E, Champion HC, Li M, Belardi D, Ren S, Rodriguez ER, Bedja D, Gabrielson KL, Wang Y, Kass DA: Chronic inhibition of cyclic GMP phosphodiesterase 5A prevents and reverses cardiac hypertrophy. Nature 2005.

11. Kukreja RC, Salloum FN, Das A: Cyclic Guanosine Monophosphate Signaling and Phosphodiesterase-5 Inhibitors in Cardioprotection. Journal Of the American College Of Cardiology 2012, 59(22):1921-1927.

12. Kuhn M: CARDIOLOGY A big-hearted molecule. Nature 2015, 519(7544):416-417.

13. Singh N, Patra S: Phosphodiesterase 9: insights from protein structure and role in therapeutics. Life sciences 2014, 106(1-2):1-11.

14. Lee YC, Martin E, Murad F: Human recombinant soluble guanylyl cyclase: expression, purification, and regulation. Proceedings of the National Academy of Sciences of the United States of America 2000, 97(20):10763-10768.

15. Li L, Haider HK, Wang L, Lu G, Ashraf M: Adenoviral short hairpin RNA therapy targeting phosphodiesterase 5a relieves cardiac remodeling and dysfunction following myocardial infarction. American Journal Of Physiology-Heart And Circulatory Physiology 2012, 302(10):H2112-H2121.

16. Lee DI, Zhu G, Sasaki T, Cho GS, Hamdani N, Holewinski R, Jo SH, Danner T, Zhang M, Rainer PP et al: Phosphodiesterase 9A controls nitric-oxide-independent cGMP and hypertrophic heart disease. Nature 2015, 519(7544):472-476.

17. Wobst J, Rumpf PM, Dang TA, Segura-Puimedon M, Erdmann J, Schunkert H: Molecular Variants of Soluble Guanylyl Cyclase Affecting Cardiovascular Risk. Circulation Journal 2015, 79(3):463-469.

18. Unwalla HJ, Li HT, Bahner I, Li MJ, Kohn D, Rossi JJ: Novel Pol II fusion promoter directs human immunodeficiency virus type 1-inducible coexpression of a short hairpin RNA and protein. Journal of virology 2006, 80(4):1863-1873.