NYMU Taipei/Help me!

From 2007.igem.org

Revision as of 13:35, 18 October 2007

Q1: Infrared Emitting Protein

• I've found parts that cantranslate green, yellow, and red fluorescence protein, but I can't find that can emit infrared rays. Can anyone help me find it? (posted by De-Ming Lin)
  • I am not sure there is a one can emit infrared light.
• What is your original idea for having the special part?
  • If we can combine glucose promoter and structural gene which can translate protein that can emit infrared ray, and put it on the finger tip, we can measure blood sugar by an non-invasive way.
  • I think it is the question about how to measure the promoter activity under glucose stimulus in our system (8/30).

Q2: Documentation of Projects

• For documentation, please all create a page for each of the five candidate projects on "My hot teams" and describe your design there. Uploading all the files of powerpoint or reference literature is very welcome! Thank you all very much for efforts of documentation ^^ Can we just document the two selected projects?
  • Reply: I just want to make the "brainstorming" part of our documentation as lush as we can ^^" I know everybody is busy, so maybe some description words with some files already done (powerpoint and literature showed on 7/13) are plenty enough ^^
• Should we have a good framework about how to record our experiments and ideas? (8/30)

Q3: Problems about Materials and Designs

• 由於沒有材料，我們無法動手進行實驗; P200、P400 Promoter 序列.......也無法確定，要火速解決! (posted by 迪璿, partially solved)
  • design has been changed. There is no P200 or P400 promoters in our current planning.
• Insulin gene的來源 (solved)
  • [http://www.dnai.org/text/mediashowcase/index2.html?id=329 A plasmid vector for expressing the human insulin gene in nonendocrine cells, 1994]
  • A method for cloning the insulin gene from total human DNA is developed on the basis of the polymerase chain reaction. Cloning of the insulin gene from the total DNA of human lymphocytes is performed to obtain a pNP-90 vector for subsequent expression of the insulin gene in nonendocrine cells for the purposes of gene therapy of diabetic angiopathies
  • [http://www.dnai.org/text/mediashowcase/index2.html?id=329 Cloning the human insulin gene, Walter Gilbert]
  • [http://highered.mcgraw-hill.com/olcweb/cgi/pluginpop.cgi?it=swf::535::535::/sites/dl/free/0072437316/120078/micro10.swf::Steps%20in%20Cloning%20a%20Gene steps in gene cloning]
  • we had the clone of human insulin from genomics center of NYMU, please see ...
• glucose sensing promoter的來源 (solved)
  • Regulated insulin serection Team Meeting 2007/07/30 (alternative method)
  • ompR binding site
    • [http://partsregistry.org/Part:BBa_R0082 Part:BBa_R0082] (15P, plate 1), pOmpC
    • [http://partsregistry.org/Part:BBa_R0083 Part:BBa_R0083] (17H, plate 1), partial pOmpC
    <li[http://partsregistry.org/Part:BBa_R0084 Part:BBa_R0084] (11H, plate 1), OmpF
• How to transport insulin?
  • From the research of Japanese scientist, we have known that fusion proteins with an alpha-hemolysin (HlyA) C-terminal signal sequence are known to be secreted by the HlyB-HlyD-TolC translocator in Escherichia coli. And in their research, a particular mutant of hlyB and hlyD can achive higher transport efficiency. (The full article is [http://www.pubmedcentral.nih.gov/articlerender.fcgi?tool=pubmed&pubmedid=15691914 here], and the source link in our site is [http://igem.ym.edu.tw/private_2007/index.php/Production_of_heterologous_proteins_in_Escherichia_coli here])
  • The twin arginine (Tat) secretion pathway allows the translocation of folded proteins across the cytoplasmic membrane of bacteria. Tat-specific signal peptides contain a characteristic amino acid motif ((S/T)RRXFLK) including two highly conserved consecutive arginine residues that are thought to be involved in the recognition of the signal peptides by the Tat translocase. (The full story can be found in [http://igem.ym.edu.tw/private_2007/index.php/Disulfide_bond_formation_in_Escherichia_coli here]. Source article is <a href="http://www.jbc.org/cgi/content/full/282/11/7903">here</a> and another article is [http://www.jbc.org/cgi/content/full/282/11/8309 here])
  • design of primers for constructing INS_A, INS_B, IDE with TAT signal peptides
• How to activate insulin?
  • Owing to that insulin requires the di-sufur binding between chain A and B to activate its activity, and E.coli seldomly forms di-sufur bounds in the cytoplasma under highly reduced environment, the solution is to mutant glutathione oxidoreductase (gor) and/or thioredoxin reductase (trxB) genes to decrease the reducing power within the membrane. See also the [http://igem.ym.edu.tw/private_2007/index.php/Image:Disulfide_reducing.jpg di-sulfur reducing pathways]. The current solution is to buy E.coli with mutants on gene gor and trxB (find out the strain we need [http://wolfson.huji.ac.il/expression/Bacterial_Strains.htm#strains-exp here]). The full story can be found [http://igem.ym.edu.tw/private_2007/index.php/Disulfide_bond_formation_in_Escherichia_coli here] (Thanks for the sharing from Dr. Chang)
  • Besides, we made the insulin to be expressed separately in alpha and beta chains only instead of the full length
• How to verify our insulin?
  • antibody of insulin
• How to inhibit our insulin?
  • IDE (insulin degrdation enzyme)
• How to prevent E.coli intakes the glucose?

Q4: how to read the plate in iGEM 2007 Kit?

• File:IGEM06DistPlateTop.jpg
• please make sure the side down has two cutting edges (triangle) to ensure the correctness of orientation

Q5: Detail sequence information needed for the parts in our design

• We need your help to find the sequence information of the parts in our design and annotate the sequence in readable format so experimental detail could be determined. Thank you!
• Parts need detail sequence information and annotation ASAP: RcsC,  EnrZ, [http://partsregistry.org/Part:BBa_R0082 Part:BBa_R0082] (15P, plate 1), [http://partsregistry.org/Part:BBa_R0083 Part:BBa_R0083] (17H, plate 1), [http://partsregistry.org/Part:BBa_R0084 Part:BBa_R0084] (11H, plate 1), insulin construct from <a class="external text" title="http://www.bcrc.firdi.org.tw/wwwbcrc/main.jsp" rel="nofollow" href="http://www.bcrc.firdi.org.tw/wwwbcrc/main.jsp">BCRC</a>
• For more information, refer to planning. If you are interested in experimental design, please feel free to write down your comment on the discussion page of planning. Thank you!

Q6: Requests for chemicals

• Anthrone for glucose-detection assay (posted by A ken)
• NdeI in part tar-EnvZ (got it!)
• SpeI in standard assembly (got it!)
• chloramphenicol (10-20ug/ml, methanol as solution, $29 for 5g) (got it!)
• human insulin
  • 林宜賢醫師說消防隊附近的川秀藥局 (2827-0702) 有賣
  • 川秀藥局 sold (8/30, 2007)
    • Humulin N [http://www.lillydiabetes.com/product/humulin_family.jsp?reqNavId=5.3 Lilly]), 中效型 (1 time/day, 混濁狀), 售價 350 NTD
    • Humulin R [http://www.lillydiabetes.com/product/humulin_family.jsp?reqNavId=5.3 Lilly]), 常規型 (2 times/day, 澄清狀), 售價 350 NTD
    • Actrapid ([http://www.novonordisk.com/diabetes/hcp/pharmaceuticals/actrapid/default.asp?bIsPro=true Novo nordisk]), 短效型 (short-acting, 澄清狀), 售價 450 NTD
    • Insulatard ([http://www.novonordisk.com/diabetes/hcp/pharmaceuticals/insulatard/default.asp Novo nordisk]), 中效型 (intermediate-acting, 混濁狀), 售價 400 NTD
  • sigma insulin

Q7: How to detect the glucose concentration in our project?

Q8: How to participate our wet lab works?

• Assembly procedure for two parts
  • STEP 1: digest full plasmid into part DNA and vector DNA (2.0 h waiting, 0.5 h operation; total 2.5 h)
  • STEP 2: gel separation for part DNA and vector DNA (1.0 h waiting, 0.5 h operation; total 1.5 h)
  • STEP 3: ligation for 2 part DNAs (3.0 h waiting, 0.5 h operation; total 3.5 h)
  • STEP 4: transformation in plate culture (16.0 h waiting, 0.5 h operation; total 16.5 h)
  • STEP 5: liquid culture to amplify the combined part DNA (16.0 h waiting, 0.0 h operation; total 16.0 h), back to STEP 1 and repeat

Q9: We need an "available restriction enzymes list and an almost depleted reagents page.

• <[http://igem.ym.edu.tw/private_2007/index.php/Available_Restriction_Enzymes Available Restriction Enzymes]
• [http://igem.ym.edu.tw/private_2007/index.php/Almost_Depleted_Reagents Almost Depleted Reagents]

Q10: How about the hotels for Jamboree 2007?

<lihttps://2007.igem.org/Jamboree/Hotels
• There are three Holiday Inn in Boston and each provides different prices!
• Holiday Inn Express Hotel & Suites Cambridge/Boston
  • Price: $149US plus applicable tax
  • Transportation to/from: 15-20 minute walk to campus

• Holiday Inn: Boston Government Center
  • Price: $179US plus applicable tax
  • Transportation to/from: 15-20 minute scenic walk across the Charles river to the Jamboree or 2 minute walk to Charles/MGH T stop (<a class="external text" title="http://www.mbta.com" rel="nofollow" _toggle_original_display="" href="http://www.mbta.com/">MBTA</a>) on the Red line (take to Kendall Square to get to Jamboree)
• Holiday Inn: Somerville
  • Price: $139US plus applicable tax
  • Transportation to/from:

Q11: About apply the US visa

• [http://www.ait.org.tw/zh/visa/niv/ http://www.ait.org.tw/zh/visa/niv/]
• Everybody who do not have a valid US visa should apply for the visa and arrange the appointment as soon as possible.
• Take special-sized photos, pay the fee, finish the online application form, and appoint the interview (Engine: 10/3 10:00)
• https://2007.igem.orglogin.cgi -> log in -> and apply an invitation letter to bring to the interview

Q12: For boys

• https://nas.immigration.gov.tw/
• Finish the DS-157 form during applying US visa

Q13: Documentation

https://2007.igem.org/Jamboree/Compete#Online_Judging

• Project Documentation on the iGEM Wiki

• Description: The team's project should be documented on the iGEM 2007 wiki site with detail enough to replicate it independently. Additional presentational information about the team - their story, the rationale for the project, failures, successes, future work, etc. - is highly encouraged. Remember that these wiki pages will be the main source of inspiration for future teams, and having good documentation on them and in the part description in the registry increases the likelihood of more teams building on your project and your parts.
• deadline: the iGEM wiki freezes 04:59 GMT Oct 27

• Part Documentation on the Registry

• Description: Document your parts well - the success of not just iGEM, but all of synthetic biology, depends on the development of well-characterized, reliable, standardized biological parts that have been designed to be simple to use and understand. The first step is complete documentation.
• deadline: the Registry freezes 04:59 GMT Oct 27

Plan to document:

• Regular meeting
• My hot teams
• Team schedule
• Help me!
• Protocols
• Materials
• Benchman
• Our Part Registry 
• Key experiments
• Construct
• Almost Depleted Reagents
• You must know!
• Paper

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