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
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
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
- Official Team Meeting on Scheduling
- Meeting with Anna-Lena and Tobias
- Introduction into R
- Tobias will give us access to their computing cluster (Group Roland Eils)
- Promoter Selection: DoOP, EnsEMBL, or UCSC?
- HUSAR account arrived
- installation of R, R editor and perl editor
- further configuration of our internal server / mediawiki
8-10-2009
- first contact with R and perl
- playing around with R and perl
- playing around with R library: Biobase
- check working on DKFZ cluster
8-11-2009
- defining programming languages: perl, R, MySQL
- retrieving first Promotersweep output files
- Meeting with Marti
- ideas for modeling
- we will have at least three colors which overlap in their spectra.
- a very nice approach will be Fuzzy Logic Modeling.
- idea 1: error checking of affinity: compare expectation to experimental results and figure out where the error is hiding
- idea 2: create&visualize fancy and fuzzy data from in silico simulation
- combine: promoter, output and graphic representation (GRAFIK!)
- next meeting with Marti: end of next week.
- extract NCBI Entrez Gene IDs with R and perl
- MAC adresses registered for bioquant network
8-12-2009
- configure perl working environment
- study structure of DoOP database
- download DoOP and load DoOP database into MySQL
8-13-2009
- trying out some DoOP queries
- download fasta sequences from UCSC gene browser (LINK)
- mapping of NCBI Entrez Gene IDs with RefSeq IDs
- configure perl working environment on Windows XP
- contact Endre Sebestyen concerning the perl module Bio-DoOP-DoOP (LINK)
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
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