Summary of issues discussed during the first meeting of the
“Sleeping Beauty” project. (28-29/11/2005)
Present: P1 [Esther Lubzens, Ora Hadas, Assaf Sukenik, Nadav Denekamp (postdoc)]; P2 [Joan Cerda, Angela Tingaud-Sequéua (post-doc)]; P3 [Stefan Hohmann,
Avi Nahmani (PhD student), Ivan Pirkov (PhD student), Cecilia Geijer (PhD
student)]: P4 [Melody Clark, Roger Worland, Gordana Gruber-Lajsic, Jelena
Pilipović (PhD student)], P5 [Richard Reinhardt, Michael Kube, Heiner Kuhl] were
present mainly on Dec 28. Full contact list is attached to this protocol.
Absent: P6 (Jens Nielson)
Notes were taken by Nadav Denekamp
During the kickoff meeting at MPI-MG (Dahlem- Berlin, Germany), presentations
were made by each partner as specified in the agenda and the following is a summary
of topics raised during the presentations and at the discussion periods.
A
presentation was made by Dr. Claus Hutchig from MPI-MG on microarrays.
Prof. Nielsen could not send a representative as the post-doc that was supposed to
work in the SB project resigned and he has not found yet a replacement.
1.
Management
1.1
Contract
All partners agreed to have a consortium project contract is preferred. Esther will send
a copy of the contract with the corrections suggested by DTU (Partner 6) to the
partners. Each institute will send in it back with comments. An accepted contract
should be signed within 6 months.
1.2
Publications
During the period until the signing of an official contract that specifies the intellectual
properties of the consortium and its partners, every one who wishes to publish
something that might be connected to the project will send an abstract to Esther that
will distribute it to all the other partners for appoval. The approval from partners
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should be done within 14 days of the e-mail specifying the request. The abstract of the
final submitted manuscript should not differ significantly from that of the abstract.
1.3
Parallel projects
The same mechanism as specified in 1.2 should apply to proposals or applications for
funding for projects on topics included within Sleeping Beauty. Anyone who whishes
to apply for funding based on ideas from this project will send an abstract to Esther
and it will be distributed the other partners and the partners should reply within 14
days.
1.4
Disputes
Disputed will be sorted within the committee of all partners.
1.5
Report's Schedule
Two reports should be submitted: after 18 months and after 36 months. Abstracts
should be prepared every 6 months and sent to Esther and Esther will distribute them
before the biennial or annual meeting. It was agreed that at the mid-project report (18
months) an evaluation and discussion will be held on the progress of the project and if
absolutely necessary the issue of extending the project for 2- 3 months (at no extra
cost) will be raised with the EC scientific officer.
1.6
Project website
A website for the project will be constructed by Stefan’s group. The web site will
contain both public and private sections.
1.7
Organization of a workshop on dormancy and publication of proceedings
A workshop on dormancy should be organized toward the end of the project (2008).
Since applications for external support through the ESF-EMBO program will have to
be submitted by May 2006 for programs in 2008, each partner should raise ideas
about the structure and dates within the next few months. A proposal for funding this
workshop must be ready before the second meeting in spring 2006. In case of a
submission of a proposal to the ESF-EMBO organization, the leading person should
be one of the European partners as Israel is not a full member of the ESF and cannot
lead an application. There are several options for the site of the workshop (1) To have
the workshop in Europe, (2) To have the workshop in Israel and Esther will seek the
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support of the Israeli Academy and other Israeli funds, including bi-national funds
(British Council; GIF; The Israel Ministry of Science). Stefan has agreed to publish
the book.
1.8 Next meetings
1.8.1 In May 2006, there will be a two days meeting in Göteborg, Sweden, organized
by Stefan (Partner 3). Each partner should send an abstract describing the
progress in the project before the meeting to Esther and it will be distributed to
all partners together with a draft of the agenda. The final agenda will be
decided by comments from all partners on the draft.
At the meeting
discussions will take place on the following topics:
-
Genomoics : types of cDNA libraries (subtractive; normalized), the
type of microarrays.
-
Proteomics: Sample preparation, 2D gel analysis.
-
Database: when to have the workshop on bioinformatics for all the
partners
Questions to Richard should be sent on April (1 month before the meeting).
The specific date will be decided by e-mail communications.
1.8.2. The annual meeting will take place in September in Cambridge. The exact
date will be decided by e-mail communication and during the biennial meeting
in Sweden.
1.8.3. Esther expressed a wish to have at least one of the following meetings in Israel,
depending on the agreement by all partners.
1.9
An internal workshop
An internal workshop should be held 1 year before the end of the project and 6
months before the general workshop
2. Sending samples to MPI-MG
2.1 According to Annex 1, MPI-MG is to provide the following platforms to partners
by the budget allocated to them in the project:
•
56k ESTs
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•
~ 14 libraries (Subtractive; normalized)
•
Microarrays: 3,000 genes /array; 40 experiments
•
Proteome profiling: 2D-gels; ~analyses of ~400 spots
Partners can discuss with Richard additional tasks on account of the budget allocated
to each partner. These requests need to be discussed well in advance.
2.2 RNA extraction
For the construction of cDNA libraries, RNA samples should be extracted from the
fresh samples by each partner. 1 g of total RNA is needed (i.e. 1 gr of fresh weight
of tissue).The extracted RNA should be checked on an agarose gel. The RNA samples
should be sent in aliquots with a picture of the agarose gel.
A protocol for RNA extraction will be sent by Richard (or posted on the website).
Samples will be sent during the next 6 months; except for the killifish samples that
will be ready sooner).
The issue for cyanobacteria was discussed separately as
Richard and Michael raised the issue that it maybe better to carry out a full genome
sequencing of this organism. The protocol of the discussions that took place between
Assaf, Ora (P1) and Richard and Michael (P5) is given separately below.
2.3 cDNA libraries
MPI-MG can make 56K EST’s (~14K per organism). This limits the number of
cDNA libraries that can be done for each organism.
The sort of cDNA libraries that would be prepared by MPI-MG will be discussed
further in the next meeting (will be held during May in Göteborg). Several options
were suggested:
(a) A library from each stage and a subtractive library.
(b) A library from each stage and a linearized library from a mixture of all stages.
2.4 Microarrays
There are two kinds of microarrays: cDNA based arrays and oligonucleotide based
arrays. Joan raised the issue that since several tens of thousands of ESTs are available
today in the USA for the killifish, a nucleotide based array maybe an advantage for
studies on the killifish. Richard prefers to make cDNA based arrays. It is preferred
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that the preparation of microarrays will be done for the all the groups at the same
time. As stated above, 40 hybridizations are budgeted (10 for each organism) and
more could be discussed with Richard in advance of printing.
2.5 Proteomics
Extraction of proteins from the samples will be done by each group. The protein
sample should be checked on a gel. A further discussion for what is needed for
proteomics will be held in the next meeting.
3. Data analysis and Database construction (WP5)
3.1 Data analysis will be done in two stages:
Stage 1: Will be done by each group. Annotation will be done by Richard. Basic data
analysis and Interpretation of species specific data will be done by each group.
Sefan’s group (Avi) will provide support to other partners. Links for bioinformatics
tools will be posted on the website of the project.
Stage 2: Melody offered to have a workshop for all partners to compare the results
from the different organisms to fulfill the requirements for WP5.
3.2 Storage of the EST’s
Richard will send the EST’s to EBI. They can keep it private for 2 years. In special
cases they may keep some genes for an extended period.
4. Minutes of a coordination meeting held at MOLGEN MPI Berlin (November
29, 1600-1730)
Present: Richard Reinhardt, Michael Kube, Ora Hads and Assaf Sukenik
Summarized by: Assaf Sukenik
The aim of the meeting was to define potential obstacles in the preparation of EST
libraries of Aphanizomenon ovalisporum, the cyanobacteria model within the Sleeping
Beauty EU NEST project.
4.1 The problems in establishment of EST library were defined and mainly stem from
the lack of adequate methodology to obtain pure mRNA from cyanobacteria. (The
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absence of 3’ polyA sequence in the mRNA requires a substantial removal step of the
rRNA fraction. The current available procedures for this step are of low efficiency
and remove only 30 to 50% of the rRNA).
4.2 The preferred direction will be a shot-gun DNA library and mass sequencing of
ca 15,000 genomic fragments (assuming fragment average size of 500 bp). Given a
genomic size of 6.4 Mb encoding for ca 5400 proteins (as found for Anabaena sp.),
this effort expect to cover 65% of the entire Aphanizomenon genome.
4.3 The proposed work has to be done with axenic culture of Aphanizomenonm
otherwise we might be dealing with unknown sequences from one or more
contaminating bacteria source.
Measure – make an effort to clean a culture to proven axenic condition.
4.4 In order to verify the status of current available cultures and the potential
interference from contaminating eubacteria, it was suggested to run preliminary study
that will screen the presence of eubacteria rRNS sequence copies versus
cyanobacteria rRNA copies.
Measure – prepare Aphanizomenon cultures: 1. A culture cultivated under
optimal conditions to a late exponential growth phase, 2. A culture cultivated under N
limited conditions and developed heterocysts. Harvest the culture at late exponential
phase, 3. A culture cultivated under P limiting conditions for 7 or 10 days. All
cultures will be harvested by centrifugation washed 3 times with 0.22 m filtered
sterilized fresh medium. Send to MOLGEN MPI-Berlin frozen biomass pellet.
Responsibility – Ora Hadas
4.5 The issue of 2-D protein gel was discussed and an advice was asked regarding the
preparation of protein samples for 2-D analyses that will be performed by the MPI
team. Although a direct solution was not provided it was suggested to discuss the
issue based on specific 1-D gels that will run with samples in question.
Measure – run 1-D PAGE with vegetative cells and akinetes and send visual
results to MPI team for further discussion.
Responsibility – Assaf Sukenik
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4.6 The issue of Peridinium (Dinoflagellate) genome project was briefly presented
and its relevance and importance were shortly discussed. This future project is not
connected to Sleeping Beauty NEST-EU activity and will be further discussed after
more information on Peridinium will be provided.
Measure – prepare a full description of the Peridinium issue and its relevancy to
Lake Kinneret and water issues in Israel. Include literature search on genomic
data of related organisms
Responsibility – Assaf Sukenik
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5. Sleeping Beauty contact list
Partner
Name
email
Partner 1
Esther Lubzens
esther@ocean.org.il
(NIO)
Other
elubzens@hotmail.com
Nadav Denekamp
nadavd@ocean.org.il
nadavden (skype)
nadav_den@walla.co.il
Irena Pekarski
irena@ocean.org.il
Alisa Hadani
aliza@ocean.org.il
Partner 1
Assaf Sukenik
assaf@ocean.org.il
(KLL)
Ora Hadas
orah@ocean.org.il
ohadas@umassd.edu
Partner 2
Joan Cerdà
jcerda@icm.csic.es
(IRTA)
Angela Tingaud-Sequeira tingaud@icm.csic.es
Partner 3
Stefan Hohmann
hohmann@gmm.gu.se
(UGOT)
Cecilia Geijer
cecilia.geijer@gmm.gu.se
Ivan Pirkov
ivan.pirkov@gmm.gu.se
Abraham Nahmany
abraham.nahmany@gmm.gu.se
Partner 4
Melody Clark
mscl@bas.ac.uk
(BAS)
Roger Worland
mrwo@bas.ac.uk
Gordana Grubor-Lajsic
gordanagl@ib.ns.ac.yu
Jelena Pilipović
jepi@bas.ac.uk
pilipovic_j@yahoo.com
Partner 5
Richard Reinhardt
(MPI-MG) Michael Kube
rr@molgen.mpg.de
kube@molgen.mpg.de
Heiner Kuhl
kuhl@molgen.mpg.de
Partner 6
Jens Nielsen
jn@biocentrum.dtu.dk
(DTU)
Trine Bro
trb@biocentrum.dtu.dk
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avi_nahmany (skype)