Team:Heidelberg/Notebook modeling

From 2009.igem.org

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* first contact with several databases: EmsEMBL, Compara, cisRED, DoOP, TiProD, contra (LINKS)
* first contact with several databases: EmsEMBL, Compara, cisRED, DoOP, TiProD, contra (LINKS)
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== 8-6-2009 ==
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=== 8-6-2009 ===
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*Miniprep of p32, p33, p34, p35, p36, p37, p38, p39, p40, p41, p42, p43
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* Meeting with Oliver Pelz
 +
** defining workflow with PromoterSweep, Matrix Profile Search and introduction into different Motif Discovery Algorithms
 +
 
 +
* installation of NX server for access onto internal server from Windows
 +
* configure developing environment (printing from Linux, configure Mediawiki)
 +
* defining basic concept of database construction
 +
** we select annotated promoter sequences in DoOP
 +
** we make a selection of pathway of interest using KEGG
 +
** narrow down number of target promoter sequences <10000.
== 8-7-2009 ==
== 8-7-2009 ==

Revision as of 23:12, 18 October 2009

Notebook HEARTBEAT

Welcome to the notebook of the HEARTBEAT (Heidelberg Artificial Transcription Factor Binding Sites Assembly and Engineering Tool) project. This notebook comprises the work on three sublanes: HEARTBEAT database (DB), HEARTBEAT graphical user interface (GUI) and HEARTBEAT fuzzy modeling (FN) as well as some additional work on logo as well as wiki design. Have fun!


Contents

  • August
  • September
  • October
Week Days
32 8-3-2009 8-4-2009 8-5-2009 8-6-2009 8-7-2009 - -
33 - - 8-12-2009 - 8-14-2009 - -
34 8-17-2009 8-18-2009 8-19-2009 8-20-2009 8-21-2009 - -

7-27-2009

  • Meeting with Oliver Pelz
    • Discuss general ideas of our Database Structure and Content
    • An introduction into PromoterSweep (LINK). PromoterSweep screens a given sequence for conserved regions giving us consensus sequences and moreover screens them for TFBS by using database search (TRANSFAC, Jasper) (LINK)
    • Our new database should contain following informations: promoter sequence, TFs, TFBS, position of TFBS, number of binding TFBS, "host organism"
    • We decide to choose MySQL as a appropiate language solving this challenge which allows us also a graphical representation of the database on the web later.
    • GUI on wiki: which language? php? javascript?
    • Problems: access to PromoterSweep (Husar Bioinformatics Group, DKFZ), choice of Promoter Database (DoOP, UCSC, EnsEMBL) (LINK)
  • aim: create database until end of August

AUGUST

8-3-2009

  • First contact with MySQL
  • Start making an overview of other team's projects
  • Configuring our Virtual Server

8-4-2009

  • Official Team Meeting (LINK) @ BQ seminar room 43: preparaing presentation & writing meeting report
  • Start installing developing environment on our internal server
    • GNOME
    • Mediawiki

8-5-2009

  • Meeting with Tobias Bauer & Anna-Lena Kranz (Theoretical Bioinformatics, DKFZ) @ TP3, DKFZ
    • Integrating ideas of PromoterSweep, Transfac as well as DoOP/CisRED
    • select "interesting" TFs (e.g. HIF, NFkB, c-myc, p53) for Wetlab
    • select "interesting" pathways (e.g. cell cycle, inflammation, metabolism etc)
    • future experimental validation: ChIP-on-Chip
      • for this we need a TFBS-free sequence
    • idea: plot histogram of TFBS relative to TSS
      • problem: choice of sequence: upstream only? inculde downstream?
    • new programming language: R and perl
    • next meeting: Friday after team meeting
  • Meeting with Karl-Heinz Glatting (HUSAR, DKFZ) @ TP3, DKFZ
    • An introduction into PromoterSweep
    • Structure and analysis principles of PromoterSweep
    • Output is stored in an XML file. This means we have to parse the xml code.
    • Oliver Pelz will give help for us in programming
  • Protocol of the meeting can be downloaded from here.
  • Start working with MySQL
  • request UNIX/HUSAR/HPC access at DKFZ (Nao)
  • first contact with several databases: EmsEMBL, Compara, cisRED, DoOP, TiProD, contra (LINKS)

8-6-2009

  • Meeting with Oliver Pelz
    • defining workflow with PromoterSweep, Matrix Profile Search and introduction into different Motif Discovery Algorithms
  • installation of NX server for access onto internal server from Windows
  • configure developing environment (printing from Linux, configure Mediawiki)
  • defining basic concept of database construction
    • we select annotated promoter sequences in DoOP
    • we make a selection of pathway of interest using KEGG
    • narrow down number of target promoter sequences <10000.

8-7-2009

  • Ordered primers for: GPI-attachement signal, Ras1b, YFP, CFP, GFP, mi

8-12-2009

  • ordered primers for the mutagenesis PCR of the fluorescent proteins


Plasmid restriction site mutagenesis position
p41 PstI 1257
p43 PstI 1001
p37-Ras PstI 840
p37 NheI/EcoRI/PstI 18/789/840
p33 NheI/PstI/PstI/EcoRI 34/259/799/805
p35 NheI/NheI/PstI 28/67/148

8-14-2009

  • two mutagenesis PCR's were done (p33g259a and p37a789g)

8-17-2009

  • the other mutagenesis PCR's were done
  • and all mutagenesis PCR products were transformed into DH5alpha bacterial cells

8-18-2009

  • no colony because of problems with the kanamycin plates
  • all mutagenesis PCR's were repeated
  • started to test the ligation orientation of inserts into BBb plasmids:

- Digested p31 and a Jet insert with NheI and SpeI. -Ligated both together for 3 hrs at RT. (insert:vector= 1:1, 3:1, 1:3) -Transformed bacteria with ligate and plated them on Amp plates. -Repeated mutagenesis PCR again for (p33, p35, p37, p37-Ras, p41, p43)

8-19-2009

  • Ligation worked for all 3 plates. Picked 6 colonies for minipreping from each plate for analysis.
  • PCR amplification of each of GPI-attachement signal, EBFP alone, NLS alone and EBFP together with NLS.
  • Ran EBFP/NLS amplicons on gel and the run seemed to work.


eBFP/NLS PCR amplification

  • GPI-attachement signal was amplified overnight.

8-20-2009

  • Restriction digestion of ligation products after minipreping.
  • Ran PCR products for GPI on gel--> nothing!:(
  • Restriction digestion showed only efficiency of 5% :( (only 1/18 digests showed insertion in the opposing direction)

8-21-2009

  • new mutagenesis PCR (without kit)

8-22-2009

  • Insert Amplification of mitoneet-eGFP by PCR

8-24-2009

  • Restriction digest of mutagenized Plasmids (PstI) and analysis on gel
  • Amplified inserts were gel-purificated


  • What worked: eBFP, eBFP+NLS, eBFP_kozak, eBFP+NLS_kozak, eGFP, eGFP_kozak
  • What didn't: NLS, NLS_kozak, eGFP+mitomeet, mitomeet, eGFP+mitomeet_kozak, mitomeet_kozak,

8-26-2009

BBBing of Insertsequences

  • PCR of cherry, cherry_myrpalm, myrpalm, NLS with kozak Primers to amplify cherry_kozak, cherry_myrpalm_kozak, myrpalm_kozak, NLS_kozak
  • Restriction with NheI and SpeI of localisationsequences and Flourophores, Restricted Plasmid was provided by Synthetic Promoter Group and digested with SAP
  • Ligation with p31
  • Transformation in DH5alpha with ligated Plasmids
  • Outplating of Transformed cells on Amp-plates

8-27-2009

  • Ligation and Transformation did not work (no colonies, except of two on the NLS )
  • New PCR with flourophores and localisationsequences, to get higher amounts
  • GEl purification of: eGFP, eGFP_kozak, eBFP, eBFP_NLS, eBFP_kozak, eBFP_NLS_kozak, NLS_kozak, cherry, cherry_myrpalm, myrpalm, cherry_kozak, cherry_myrpalm_kozak, myrpalm_kozak

8-28-2009

BBBing of Insertsequences2.0

  • Restrictiondigest of flourophores and localisationsequences with SpeI and NheI (1 h, Buffer 2, BSA)
  • Restrictiondigest of p49 with SpeI and NheI (1 h, Buffer 2, BSA) and SAP (30 min), purification
  • Nanodrop of digest shows no DNA inside of the samples -- purification was maybe unsuccessful

8-29-2009

BBBing of Insertsequences2.1

  • Restrictiondigest of flourophores and localisationsequences with SpeI and NheI (1 h, Buffer 2, BSA)
  • Restrictiondigest of p49 with SpeI and NheI (1 h, Buffer 2, BSA) and SAP (30 min), purification

8-31-2009

BBBing of Insertsequences2.1 (part 2)

  • Ligation of Insertsequences with restricted p49
  • Transformation
  • Outplating -> Wrong resistance