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

• July

• August

• September

• October

July

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

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August

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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

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
• writing first perl program - "Hi there"

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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
  • 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
• 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

8-14-2009

• parse UCSC fasta sequences according to our selection
• write parsed sequences into multifasta format
• start PromoterSweep Analysis over Weekend

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8-18-2009

Tim, Stephen, ab hier müsst ihr eure Sachen selber eintragen!

• study outputfile of PromoterSweep. check out general structure and pick up useful information.
• result is grouped in: General Info, Best Genomic Mapping, Promoter DB Search Result, Graphical Overview, Combined Binding Sites, TSS and Exon Info, Profile Matrices and Generated Output Files.
• upon selection, sections of interest will be collected and made ready for entry into MySQL DB
• discuss table structure of our database

• How should our database be called? - Brainstorming -
  • SHOULD contain: iGEM, Transcription Factor, Binding Site, Promoter, synthetic biology, Heidelberg
  • MAY contain: position, heartbeat, prediction, assembly, eukaryotes
  • and still more keywords to come
• establishing local@host access to mysql

8-19-2009

• parse Promotersweep xml file into tab-separated text file
  • the text file should contain: RefSeq ID, TF name, TFBS position, TF motif sequence, TFBS Quality, TSS, Entrez ID, EnsEMBL ID, further gene description.
  • this provided us with several programming problems concerning working with multiple arrays, hashes and their combinations (arrays of hashes, hashes of hashes, etc.) thus
• studying structure and basic concepts of hash & key
• including parsed data into mysql database

8-20-2009

• pre-decision for our table-structure
  • Table: Main_Info
    • RefSeq ID, TF, TF motif start & end position, TFBS motif score, TFBS quality, TSS database info
  • Table: Gene_Info
    • Ensembl_ID, Gene Symbol, Gene Description.
  • we go for the RefSeq ID to be the key connecting these two tables.

8-21-2009

• update script for parsing the Promotersweep output files due to unexpected errors
• we forgot to include "weak" as a category for the TFBS quality - added!
• PromoterSweep result contains information about TSS derived from different promoter databases. On which should we rely, if they differ from each other?
  • We set our highest priority to DoOP database since they show a good accordance within the RefseqID results when compared to other databases (e.g. DBTSS).

• order [http://www.mathworks.com/| Matlab] iGEM licence

• search for a tool to use MySQL in R programming environment
• wiki: write an short article about the German Cancer Research Center (DKFZ)

• Meeting with Anna-Lena: once we established our database... then
  • two strategies:
    • manually select interesting transcription factors and analyse them using database queries
    • plot histograms of TFBS occurance within the target promoter sequence (TSS - 1000bp upstream) for each TF and make systematic analysis
  • we go for both!
  • idea for the future: we can analyze combinatorial appearance of distinct TF pairs

• We have a name for our database - we call it -

- wait for it -

HEARTBEAT database (Heidelberg Artificial Transcription Factor Binding Site Engineering and Assembly Tool)


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8-24-2009

• Meeting with Marti: defining output modeling strategies
  • "exclusive promoters"
    • a model for predicting the behaviour of activation of one, two, three... promoters at the same time.
    • the potential of this model lies in the possibility to model single as well as many pathways in combination and even check for synergistic effects
    • modeling logic: quantitative ODE VS. quantitative & qualitative fuzzy logic
  • "error checking"
    • what to capture/measure: affinity of transcription factor binding to DNA
      • calculate score / reliabilty
      • phenotypic measurement
    • if we have time in the end: model/experiment optimization by wetlab-drylab-rounds (GRAFIK)
    • if we do not have much time: figure out where is catch
  • modeling layers & final visualization
    • (i) capture affinity - (ii) model gene expression - (iii) pathway activity - (iv) fancy visualization (Mathworks Simulink?)
    • plot: time course, dynamic affinity
    • keep in mind the possible high amount of False Positives using promoter search/analysis

8-25-2009

• official Team Meeting also with Mr. Kai Ludwig (LANGE + PFLANZ) as guest for Logo / Title Claim discussion

• so far we have 1753 promoter sequences analyzed by PromoterSweep!

• Meeting with Daniela (Nao): Cell Profiler for capturing biological images & data analysis based on MATLAB

• working with R module RMySQL for using the pipeline between R and MySQL
• create a list of useful RMySQL commands

8-26-2009

• Workflow for plotting histogram - workflow (SOURCE CODE/S?)
  • make MySQL query using R
  • make list of TFs, avoid duplicates using perl
  • pick up each TF (perl/R) and plot histogram (R)

• create MySQL command list including combinatorial queries

8-27-2009

• check HEARTBEAT DB for duplicate entries
• how should we plot the histogram?
  • (a) histogram - how "wide" should be each bin? 100bp? 50bp? 20bp?
  • (b) plot probability density
• study Transfac PWM (position weight matrices) for
  • difference in consensus sequences (also ask Anna-Lena)
  • different PWM types (vertebrates, plant, insect, fungi, bacteria, nematodes...)
  • positive control: when histograms are generated and plotted, check distribution of Sp1

• so far we have 3640 promoter sequences "sweeped"!

• access from R to mysql at the local@host server established

8-28-2009

• dealing with perl - introduce transition of variables between perl and R

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8-31-2009

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September

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9-1-2009

• derive transcription factor data using R and MySQL
• plot HEARTBEAT TF hit distribution as histograms & density functions for different PWM subsets (all, vertebrates only, single matrices and joined TFs)
• further completion of the database

9-2-2009

• discussion on how to make statistical studies on our gained distributions
  • ideas: define maximum and variance -> Nao
• look for motif sequences -> Tim

• we have 4476 sequences analysed by Promotersweep so far!
  • but we are expecting 4700 sequences - check missing ones!

9-3-2009

• internal team meeting: Tim, Lars, Stephen, Nao
  • select especially interesting TFs
    • criteria: (a) good hits in our distributions; (b) easy experimental handling
    • we go for HIF, SREBP and VDR to analyse and make synthetic promoter design
• Transfac PWM: there are some annotaion inconveniences of some matrices
• which "spacer" sequences should we use in order to generate TFBS free sequece parts

• rational design of synthetic promoters
  • Tim: SREBP, Nao: VDR
  • both go for a total number of 10 sequences
  • strategies:
    • single TFs: search for density maxima
    • check combinatorial appearance and design promoter sequences with multiple binding TFs
  • use spacer sequences generated by Lars and check for TFBS using Transfac
  • sequence length: max. 1000bp

• back-up idea: if synthesis does not work for a long (~1000bp) sequence then try to work out a protocol for a two-step promoter synthesis combining one empty (TFBS free) sequence with another which consists of many TF and activator binding sites.

9-4-2009

• work with Transfac PWM: structure, description, and using consensus sequence
• write script to get the ID's and frequencies for all co-occuring TFBS of VDR and SREBP
• write script for generating consensus sequence based on Transfac PWM and replacing ambiguity code with A, C, G or T

  Getconsensus.pl, MakeConsensus.pl

• Wiki Meeting (Nao)
  • Logo choice & modification
  • choose header pics
  • navigation layout
  • develop a catchy, cool homepage

9-5-2009

• Meeting with Tim, design synthetic promoter sequences
• check spacer sequence (200bp) for TFBS: one TFBS found; remove it by cutting and shortening the sequence to 190bp)
• Kid3 is a repressor!

9-6-2009

• design more synthetic promoter sequences by manual iteration process which consists of (i) TFBS check and (ii) TFBS removal & filling up random sequence

• aim: creation of an automatic designing tool for synthetic promoters which include sequence design, transfac search as well as filling the sequence up with spacer sequences.

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9-7-2009

• check designed sequences for restriction sites

  CheckRestrictionsites.pl

• finish creating sequences
• consider CMV core promoter into the calculation of the relative position of TFBS to the TSS
• create sequences for negative control
  • pure TFBS free sequence
  • sequences with TFBS at minima of the density function
• checking for all sequences for further binding sites with the Transfac match tool

9-8-2009

• check restriction sites for reverse complementary strand
• add flanking sites with restriction sites and spacer nucleotides to our designed sequences
• is there any possibility to automatize Transfac queries?
• work with combined / joined MySQL query structures
• or solve this process by simply writing new temporary tables?

• workflow summary (short) for manual designing of a synthetic promoter:
  • (A) use random sequence
  • (B) check TF-matrices
  • (C) validate TFs (mouse? human? repressor?)
  • (D) check Transfac and restriction sites

• Phone conference with Kai Ludwig, Logo & Web Design (Nao)

• official Team Meeting

• wiki closure on Oct 21st!

9-9-2009

• modify synthetic promoter sequences to be ready for ordering
• Sweep more promoter sequences using Promotersweep
• start Modeling
• revise and improve HEARTBEAT
• discuss differences between PWMs

9-10-2009

• still modifying synthetic sequences to be ready for shipping
• we have altogether 25 designed promoter sequences!

9-11-2009

• Software Meeting (Stephen, Tim, Nao)
  • compartibility with mediawiki: HTML, perl, php, R, java?
  • GUI design
    • simple interface: single TF, auxiliary TFs, #TFBS, sequence length
    • "interactive": multiple TF, choosing auxiliary TFs, additional information (see Eukaryopedia), density function plot & histogram
    • "hyper-interactive" step-by-step design & creation

• Modeling Meeting with Marti and Anna-Lena (Tim, Nao)
  • aim: fancy visualization to show expectation & prediction providing pathway insights
  • TODO/QUESTIONS
    • what is the stimulus? collect possible inputs!
    • measurable outcome: experiments & pathways
    • quality of synthetic sequence: error checking
      • we need to define the quality of our sequences
  • LEVELS of modeling
    • (1) DNA (2) expression/transcriptional activity (3) output
    • each with corresponding measurement

• general modeling scheme: input - "What we are affecting" - possible outcomes
• how? We use fuzzy logic

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9-14-2009

• collect input for inducing the system (e.g. p53: CPT, Pifithrin-alpha; NFkB: TNF-alpha etc.)
• phone conference with Kai Ludwig
• learn how to include Perl code into html code
  • learn how to use embperl
  • configure apache2 server such that embperl can be interpreted
  • try to make offline use of embperl working
• try to find nice html editor for ubuntu - (seamonkey, Amaya)

9-15-2009

• create network picture for meeting tomorrow
• Logo discussion
• Read paper: Fuzzy Logic Modeling of Signaling Networks (Aldridge 2009)
• learn data management of virtual server
• get an overview about the apache2 file and security system

9-16-2009

• Modeling Meeting with Marti (Douaa, Tim, Nao)
  • update on available drugs/sequences
  • decide what to model: (A) error checking, and (B) differential expression?
  • use natural promoters to build up model for prediction of activity of synthetic promoters
  • Discussion of TF score
    • Transfac sequence alignment score
    • promotersweep binding site quality
    • relative position to TSS: How?
      • (A) peak width & amplitude, (B) distance to maximal peak & position, (C) number of PEAK, (D) "sliding window" and calculate area under curve, (E) #TFBS (also for comparison of different synthetic promoters)
    • biophysical affinity using TRAP
  • first model: build up either on CMV or on JeT
  • potential: integrate many stimuli -> find out crosstalks of pathways?

• TODO (meeting)
  • collect data
  • define WHAT we want to model
  • summarize available sequences
  • try to formulate IF ... THEN "sentences"
  • check MATLAB & MATLAB Fuzzy Logic Toolbox availability

9-17-2009

• internal Team Meeting
• find error.log files on the server and learn how to use it

9-18-2009

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9-20-2009

• learn how to use tag language of embperl
  • learn how to write loops with embperl
  • access of input variables in embperl -- using the %fdat hash

9-21-2009

• struggling with how to use R from embperl

9-22-2009

• Wiki Meeting (Dani, Cori, Nao)
  • install image processing tool
  • design wiki, brainstorming for possible navigation bars
• Wiki Phone Meeting with Kai Ludwig (Nao)
  • design header & presentation-master as well as team shirts

• Seminar: Martijn Luijsterburg (Karolinska Institute) - Heterochromatin Protein 1 is involved in the DNA damage response. Host: Thomas Höfer, Bioquant

9-23-2009

• Modeling Meeting with Marti, Anna-Lena (Tim, Nao)
  • contact database group (TP3)
  • statistics: characterizing peaks
    • we go for area under the curve and affinity. optionally we can choose Transfac sequence score and peak height & width
  • strategy to convince the wetlab people from the importance of modeling during the meeting on upcoming friday.
  • MATLAB license?
  • logical gates: try to start creating model topology after Friday

• Presentation: Marti Bernado Faura (Bioquant, University of Heidelberg): Data-driven Fuzzy Logic modeling of Programmed Cell Death
  • intro into fuzzy logic
  • system development & work flow of fuzzy logic
  • fuzzy inference & model prediction
  • model types: MISO / MIMO

• Wrap-up meeting: Team HEARTBEAT (Tim, Nao)
  • split up computational work into three tracks: HEARTBEAT DB, HEARTBEAT GUI and modeling
    • database: documentation (until Oct 18), peak characterization, calculate absolute density function
    • GUI: based on embperl, design according to our new wiki
    • modeling: MATLAB license, collect sequences & input data, develop network model, include pathways

• literature work

9-24-2009

• prepare slides for meeting tomorrow
• pathway search: TNF-alpha/NFkB, VDR, SREBP and crosstalks. NFkB has a lot of pathway crosstalks, while SREBP and VDR show a interesting connection. Upon induction, SREBP activates VDR.

9-25-2009

• Team Meeting (Wetlab, Nao)
  • short progress report of all of us
  • modeling: discussing scheme, modeling elements and strategies

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9-28-2009

• Wiki Phone Meeting with Kai Ludwig (Nao)

9-29-2009

• designed synthetic promoters (HB_0001 - HB_0025) will be joined to CMV core promoter since JeT core promoter contains a Sp1 site in it. All other sequences (random synthesized, e.g.) are coupled with JeT core promoter.
• literature studies on combinatorial cis-regulation as well as on modelig of the lambda-switch
• prepare slides for the next modeling meeting

9-30-2009

• Wiki Meeting (Dani, Nao)
• MATLAB license order (Jens)
• postpone Yara meeting (Wetlab, Tim)

• got sequences from Lars
• got qRT-PCR setup from Chenchen

• Modeling Meeting with Marti & Anna-Lena (Tim, Nao)
  • still need to collect FACS and microscopy results
  • discuss our network prediction model using TNF-alpha as aｎ example
  • maybe we can use the lambda switch paper as a good starting point for our modeling

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October

10-1-2009

• Wiki & Presentation Meeting with Dani (Nao)

10-2-2009

• some wiki work

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10-5-2009

10-6-2009

• Internal Team Meeting
  • check out number and measurement plans of randomly assembled synthetic promoters (5x NFkB, 5x p53, 2x pPARg, 2x SREBP)

• Wiki Meeting (Corinna, Daniela, Nao)
  • discuss design of the top page and possible features
  • try out CSS design

10-7-2009

• Wiki Design (Nao)
• Wiki Phone Meeting with Kai Ludwig (Nao)

• MATLAB has arrived!
• literature work

• Wetlab Meeting: progress report on measurement of random assembled synthetic promoters
• make thoughts about the whole storyboard of our presentation at the jamboree

10-8-2009

• Short Meeting with Roland
• image processing work for wiki

10-9-2009

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10-12-2009

10-13-2009

• Measurement discussion with Lars: REU/RMPU, defining equations for mammalian systems
• literature work on PoPS paper (Kelly JR et al.) and apply their equations

• Marti Modeling Meeting (Anna-Lena, Tim, Nao)
  • Journal Club (Tim, Nao)
  • summary of meeting from Team Meeting from last thursday

• Marti: start modeling using MATLAB and Fuzzy Logic Toolbox (FLT), playing around with FLT and tutorial

10-14-2009

Nao

• develop first test fuzzy inference system (FIS) for testing
• Marti Modeling Meeting, specify model topology
• collect data: FACS (Cori), Microscopy (Hannah), Sequence & TECAN (Lars)
• start calculating position score using R
• translating project abstract

10-15-2009

Nao

• calculate affinity score using TRAP (Anna-Lena)
• collect ideas for integrating TFwise scores in order to calculate final position/affinity score for one sequence: median, mean, maximum, weighted mean?
• all data analysis is stored in three sheets (SequenceAnalysis, ResultSummary and CalculateTRAP)
• from now on we concentrate on FACS measurements because they are the most reliable ones (TECAN used only for scanning)
• fill up TRAP data with missing transcription factors

10-16-2009

Nao

• Anna-Lena Meeting: discuss how to integrate sequence scores
• get & check p53, pPARg and random SREBP sequences
• go through FACS results
• add HEARTBEAT sequences for data analysis
• modeling documentation
• parsing experimental setups for modeling use
• Chenchen qRT-PCR results

• define possible modeling layers
  • first layer: input
    • drug type, pathway, drug mode of action, drug concentration, targeted cells, incubation time
    • sequence type, position score, affinity score
    • we choose position & affinity score, sequence type and the presence of stimulation. Time as well as different concentration (unfortunately no data available) can be added in future
  • second layer: promoters
    • 6 constitutives, 3 standards, 6 inducible available
    • data analysis narrows this to 5 constitutives, 3 standards and 4 inducible
    • HEARTBEAT sequences have to be measured a.s.a.p.

• Marti Modeling Meeting
  • try to define some fuzzy rules
  • we assume better binding -> better expression
  • define membership functions
  • start modeling with NFkB results

All

• Internal Team Meeting
  • reminder: wiki task, wiki to do
• Official Team Meeting

10-17-2009

• final decision: we go for maximum of position and affinity score
• added HB sequences for data analysis table; as soon as results are there we can model designed synthetic promoters
• define shape of membership functions
• literature search for missing activity values?
• still TODO: check out p53 results since the p53-NFkB crosstalk is really interesting!

10-18-2009

Nao

• SREBP/VDR paper arrived
• finish data analysis
• study & playing around with MATLAB FLT, programming from both FLT GUI and MATLAB command line
• define our work to be (i) error checking and (ii) exclusive pathway modeling
• high potential of this model lies in its plug'n'play structrue, with a high capacity of integrating more inputs, outputs and also the middle layer (promoter diversity)

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10-19-2009

Nao

• define final network structure
• wiki work
• reading RFC documentation and correction
• we call this project HEARTBEAT fuzzy network (FN)
• HB FN documentation and first results!
  • creating two fuzzy controllers: inducible NFkB and constitutive
• how do we integrate the data? combine via Simulink!

10-20-2009

• Creating, developing, integrating and combining fuzzy network modeling (MATLAB, Simulink)
• first analysis of HB sequences
• HEARTBEAT FN documentation

10-21-2009

10-22-2009

• FROZEN WIKI!!!!

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