Team:Freiburg bioware/Notebook/May

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02.05 13-20.30o'clock, R&uuml;diger

datamining and investigation about DNA und FOKI. Paper Miller07, Wah1998.

3d analysis and pictures taken from pymol.

Alignment of 2BamHI and 2FOKI to get DNA in proper position. However it is still not perfekt,

but gives a reasonable insight into distances we need for the linker and where we can position the oligo

05th may, 11-13o'clock R&uuml;diger Sponsoringmeeting with Timo, Anika, R&uuml;diger

to lazy to translate already taken notes....

Uni-interne zeitschriften zuspammen

-nachwuchsf&ouml;rderung

-lernen in der praxis

-kooperatives lernen

-gruppenmanagement

firmenzeitschriften finden und zuspammen

Sponsoren tour

fernsehen??!

radio

Mentorenprogramm

to do:

argumentenliste (schleimig)

-ziele

-selbstst&auml;ndig arbeiten

-eigenes projekt von planung bis zur vollendung, sowas gibt es sonst nirgends. Einblick in zukunft unseres

Forscherlebens.

neues universelles werkzeug f&uuml;r synthetische biologen: soll laboralltag revolutionieren.

2Projekte:

Entwicklung eines universellen Schneidewerkzeuges: Man k&ouml;nnte es mit einem Schraubenzieher vergleichen,

auf den man alle arten von Bits aufsetzen kann. Jede Art von Schraube ist mit einem einzigen ,statt,

wie bisher mit hunderten verschiedenen Schraubenziehern herausdrehbar.

Es soll neue opensource Software f&uuml;r iGEM entwickelt werden, das die komplete Arbeit mit den BiobrickTM

Standard Parts in einer einzigen Anwendung zusammenf&uuml;hrt. Dies beinhaltet das Suchen und runterladen der

Sequenzen, sowie die Zusammenstellung und Bearbeitung, bis hin zur Ver&ouml;ffentlichung der Sequenzen in Form eines

neuen Biobrick Parts.

Man kann sich das wie Lego basteln vorstellen: Beispielsweise man m&ouml;chte ein Haus aus Lego bauen.

Dazu sucht man sich alle ben&ouml;tigten Steine zusammen. Aber bevor man anf&auml;ngt wild drauf los zu basteln,

zieht man das Programm zu Rate und spielt alle Varianten am Computer vorher durch. Das Programm zeigt einem

dabei mittels verst&auml;ndlicher Grafiken, was machbar und sinnvoll ist und druckt am Ende eine fertige Bauanleitung aus.

Und da das Programm der Open-source Idee verpflichtet ist, kommen alle Anleitung f&uuml;r alle zur freien

Verf&uuml;gung ins WWW.

-programm

05th may, 17o'clock R&uuml;diger 17o'clock, Meeting the Captain(Igloi) with Dieter and R&uuml;diger. Captain tells us about how to link PNA with Protein via Ni/Co and His tag. same could be applied to link ologo and protein. dna needs to hybridize with around 30 bases oligo to properly build up double helix. 07th may, 14-18o'clock R&uuml;diger Dicussing FokI Sequence and further strategy with Laura, Gerrit Dieter and Kristian.

Companys to buy Genes from:

-Geneart

-Mr.gene(low budget version without support)

-itelichon or sth like that

-DNA 2.0 (expensive but can demand adequate features of vector)

Glenn research Bioscience offers modified nucleotides for our oligos

Vektor we should create/order now:


 * must not contain IGEM restriction sites


 * 1FokI, active cleavage sitewe need to check literature about that.

Possible experiment with original cys containg fokI:

add iodoacetamide to it and look if it gets acetylated and if it still cleaves.


 * 1FokI without cleavage Aminoacids


 * replacing cys by ser (first

Possible Tags (will be investigated later in more detail):


 * Fluoreszin &amp; Digoxigenin ? coupled to oligo


 * Lipocalin can bind to these specufically


 * does the Captain have Ni/Co for Oligo synthesis?


 * or maybe Bannwarth ? ?

Program for Vector handling: Gentle

08th may, 17.15-18.30o'clock Caro Dicussing RAG1/RAG2 DNA cleavage mechanism with Caro, Manu, Gerrit, Hannes, Kristian, Cristoph.

Question discussed:


 * Which kind and how many domains have RAG1/2?


 * What are the cleavage mechanims and the interaction of theese domains?

eliminated?
 * What is the essential part of theese proteins and what can be

<b>Next meeting for RAG1/RAG2 and Recommbineering group: Monday, 11.may, 17.30 at kristians lab</b>

08th may, 13:30-19:30o'clock Laura Planning the design for two vectors including one different FokI-heterodimer each with R&uuml;diger, Dieter and Laura

Procedures for both vectors:

of the chromosomal DNA of Flavobacterium okeanokoites [http://www.ncbi.nlm.nih.gov/nuccore/148723?ordinalpos=1&amp;itool=EntrezSystem2.PEntrez.Sequence.Sequence_ResultsPanel.Sequence_RVDocSum F.okeanokoites fokIR and fokIM genes]
 * extract coding region of Fok from the restriction-modification genes


 * delete the first 1158 nucleotides/386 aa (recognition domain)


 * switch Cystein 541/463-465 to Ser (TGT-&gt;TCT)

Modifications of the single vectors to introduce heterodimeric modifications according to [[Media:Miller_J,_Rebar_E_Nature_biotech_2007_25_778.pdf&lrm;]]:

Modifications of the first vector (catalytic active heterodimer)

-heterodimeric aminio acids
 * switch Glutamate 490/310-312 to Lysin (GAA-&gt;AAA)


 * switch isoleucin 538/454-456 to Lysin (ATC-&gt;AAA)

Modifications of the second vector (catalytic inactive heterodimer)

-heterodimeric amino acids
 * switch Glutamin 486/298-300 to Glutamate (CAA-&gt;GAA)


 * switch Isoleucin 499/337-339 to Leucin (ATC-&gt;CTG)

-catalytic amino acids
 * switch Aspartate 450/190-192 to Alanin (GAC-&gt;GCG)


 * switch Aspartate 467/243-245 to Alanin (GAT-&gt;GCG)

Annotations: literature which correspond to the codons 463-465 in our vector.
 * The notation e.g. for Cystein 541/463-465 means the amino acid 541 in

from H&eacute;naut and Danchin. [http://www.faculty.ucr.edu/~mmaduro/codonusage/codontable.htm E.coli Codon Usage]
 * For exchanging the amino acids we used the Codon usage table in E.coli

Next meeting: Monday, 11th may, 16pm at kristians lab

11th may, 17.30-19.30o'clock Max Attendees: Hannes, Caro, Manuel, Gerrit, Christoph, Max

RAG: * Discussion about different domains. nicking.
 * If in RAG1 R855 and K856 are exchanged there is no hairpin but still

function and ion -induced hydroxy-cleavage.
 * RAG1: - cleavage active sites: D600,D708,E962 provides metalbinding

residues each)
 * RAG2: -The core is characterised by a six repeats of a kelch motif(50

- each repead forms a four-stranded twisted antiparallel beta-sheet(six bladed propeller). This structure takes part in protein protein binding.

- T490 phosphorylation site

- aa 352-410 acidic portion

-aa 420-480 is a Cys-His rich reagion similar to homodomains of zink fingers

-R73A, K118A/K119A Hairpin-opening

- RAG2 binds RAG1 and DNA

works?</b> Large parts can be deleted without loosing recombination activity. The full length of mouse RAG1 is 1040 amino acids. Aminoacids 384-1008 are required. Mouse RAG2 has about 527 aa. Only the first 383(387)are required. In presence of Mn2+ RAG1/2 efficiently cuts an RSS(recombination signal sequence) in a DNA fragment to yield blunt 5&acute; phosphorylated signal ends and hairpin coding ends that retain the full coding sequence. * RAG may be quite difficult to be made suitable for our purpose. out as not realizable.
 * <b>What can be taken away from the protein so that it still
 * We decided to look for another suiting enzyme just in case RAG turns

 Next meeting: Thursday, 05/14/09, 12.30 @ Zoo-Caf&eacute;

11th may, 16:00-20:30o'clock Laura Planning the design for two vectors with R&uuml;diger, Dieter and Laura

Execution of the planned modifications with the program Gentle

To avoid restriction sites of the restriction enzymes used by iGEM:


 * switch Isoleucin 109-111 to Isoleucin (ATT-&gt;ATC)


 * switch Alanine 520-522 to Alanine (GCC-&gt;GCG)

sequences of the completely edited active FOKI heterodimer:

-amino acid sequence: KSELEEKKSELRHKLKYVPHEYIELIEIARNSTQDRILEMKVMEFFMKVYGYRGKHLGGSRKPDGAIYTVGSPIDYGVIVDTKAYSGGYNLPIGQADEMQRYVKENQTRNKHINPNEWWKVYPSSVTEFKFLFVSGHFKGNYKAQLTRLNHKTNSNGAVLSVEELLIGGEMIKAGTLTLEEVRRKFNNGEINF

-nucleotide sequence: AAAAGTGAACTGGAGGAGAAGAAATCTGAACTTCGTCATAAATTGAAATATGTGCCTCATGAATATATTGAATTAATTGAAATTGCCAGAAATTCCACTCAGGATAGAATCCTTGAAATGAAGGTAATGGAATTTTTTATGAAAGTTTATGGATATAGAGGTAAACATTTGGGTGGATCAAGGAAACCGGACGGAGCAATTTATACTGTCGGATCTCCTATTGATTACGGTGTGATCGTGGATACTAAAGCTTATAGCGGAGGTTATAATCTGCCAATTGGCCAAGCAGATGAAATGCAACGATATGTCAAAGAAAATCAAACACGAAACAAACATATCAACCCTAATGAATGGTGGAAAGTCTATCCATCTTCTGTAACGGAATTTAAGTTTTTATTTGTGAGTGGTCACTTTAAAGGAAACTACAAAGCTCAGCTTACACGATTAAATCATAAAACTAATTCTAATGGAGCTGTTCTTAGTGTAGAAGAGCTTTTAATTGGTGGAGAAATGATTAAAGCGGGCACATTAACCTTAGAGGAAGTGAGACGGAAATTTAATAACGGCGAGATAAACTTT

sequences ot the completely edited inactive FOKI heterodimer: -amino acid sequence:

KSELEEKKSELRHKLKYVPHEYIELIEIARNSTQDRILEMKVMEFFMKVYGYRGKHLGGSRKPDGAIYTVGSPIDYGVIVDTKAYSGGYNLPIGQADEMQRYVKENQTRNKHINPNEWWKVYPSSVTEFKFLFVSGHFKGNYKAQLTRLNHKTNSNGAVLSVEELLIGGEMIKAGTLTLEEVRRKFNNGEINF

-nucleotide sequence: AAAAGTGAACTGGAGGAGAAGAAATCTGAACTTCGTCATAAATTGAAATATGTGCCTCATGAATATATTGAATTAATTGAAATTGCCAGAAATTCCACTCAGGATAGAATCCTTGAAATGAAGGTAATGGAATTTTTTATGAAAGTTTATGGATATAGAGGTAAACATTTGGGTGGATCAAGGAAACCGGCGGGAGCAATTTATACTGTCGGATCTCCTATTGATTACGGTGTGATCGTGGCGACTAAAGCTTATAGCGGAGGTTATAATCTGCCAATTGGCCAAGCAGATGAAATGGAACGATATGTCGAAGAAAATCAAACACGAAACAAACATCTGAACCCTAATGAATGGTGGAAAGTCTATCCATCTTCTGTAACGGAATTTAAGTTTTTATTTGTGAGTGGTCACTTTAAAGGAAACTACAAAGCTCAGCTTACACGATTAAATCATATCACTAATTCTAATGGAGCTGTTCTTAGTGTAGAAGAGCTTTTAATTGGTGGAGAAATGATTAAAGCGGGCACATTAACCTTAGAGGAAGTGAGACGGAAATTTAATAACGGCGAGATAAACTTT

Introduction of prefix and suffix according to the Fusion Protein (Freiburg)Biobrick assembly standard



Ask for an offer for our two vectors from "Biomatik Corporation"

14th May 12:30-15:00 o'clock, Hannes Attendees: Hannes, Caro, Gerrit

RAG

- preparation of RAG-presentation for the meeting on friday

- discussion of new ideas (AloI)

15th may 2009, 13:00- 16:00 o'clock Anika Attendees: Max, Sascha, Timo, Hannes, Dieter, R&uuml;diger, Laura, Julia, Caro, Paul, Gerrit, Manuel, Kristian, Anika

Presentation of different subprojects

- FOKI

genes are ordered, each vector costs approx. 344 USD, we ordered two vectors for the beginning

- homepage

basis is written in html so that everybody can contribute to complete the page, everything in english, except the 'Vereinssatzung'in german

authors of the subitems:

Team: Caro

Project: R&uuml;diger

Publicity: Manu

iGEM Contest: Anika, Timo

iGEM e.V.: Laura

-&gt; send everything to Sascha

- software

still looking for ideas, Java seems to be a good idea, program should run without an internt connnection, a Plug-In based, we will need some biologist testing this program

- sponsoring/marketing

discussion about different possibilities to find donators

- RAG1+2

group presented their plans, for the moment it seems that FOKI has the better prospect

next meeting: Wednesday, 20th May 2009

20th may 2009, 11:00 o'clock ct. Anika Attendees: Imi, Laura, Caro, Christoph, R&uuml;diger, Sascha, Kristian, Anika

Topics:

1. Vereinsbelangen

Problem: If we are going to be an 'allgemeinn&uuml;tziger Verein' it is prohibited to make advertising for our sponsors. On our website as well as on our T- shirts.

Caro and Laura will go to the tax and legal advice of the u-Asta.

2. Sponsoring

We are going to start posting the letters to the companies beginning of next week. There will be an extra meeting for the sponsoring group which is not arranged yet.

3. Plasmidconstruction

The ordered things arrivedwhich means that the laboratory work can start. The FokI group is looking for people who have time to work in the lab for cloning.

One more thing: the website is going to put online on Sunday this week. 25th may 2009, 10:00- 17:00 o'clock Laura and Caro Attendees: Max, Sascha, Hannes, Gerrit, Anika, Christoph, Imi, Sonja, Caro, Laura

Introduction into the lab work:

Hier die Einf&uuml;hrung auf deutsch (muss ja nicht ins richtige Wiki &uuml;bernommen werden):

Unser Laborraum:

bei Handhabung mit elektrokompetenten Zellen
 * UV-Ger&auml;t auf K&uuml;hlschrank zum Reinigen der Pipetten

bench (evtl. selber gie&szlig;en)
 * fertig gegossene SDS-Gele in K&uuml;hlschrank hinter unserer


 * Kopierkarte auf Annas Platz hinter unserem Laborplatz

Sterilfiltrieren (f&uuml;r Puffer etc.) und gro&szlig;e Kolben
 * Zellkulturschrank (neben Zellkultur): Zellkulturflaschen zum

Messraum (EKTA-Raum?): Viva spins (Zentrifugeneins&auml;tze bei Proteinaufzentrifugation) im oberen Schrank; Spitzen (verschiedene Gr&ouml;&szlig;en) im unteren Schrank

PCR/Gelraum (von der T&uuml;r zw. PRC-Raum und unserem Labor gesehen):


 * Ethidiumbromidverschmutzter Bereich auf der bench

Ausgang an der zweiten T&uuml;r gr&ouml;&szlig;ere Abfalltonnen f&uuml;r EthBr (Achtung: keine Handschuhe!) und Glasbruchabfall
 * Ethidiumbromid und normaler Abfall auf der bench getrennt; auch beim

&uuml;ber Tonnen
 * Proteinpuffer und Comassief&auml;rbel&ouml;sungen in Regal

f&uuml;r Klone und Proteine im unteren Teil 3. Spalte, 1. Zeile
 * eigenes Fach im -80&deg;C K&uuml;hlschrank oben; Fach


 * Waage hinten rechts in der Ecke

rechten Seite
 * nichtexplosive und nichtgiftige Chemikalien im Schrank auf der


 * brennbare und explosive Substanzen im gelben Schrank an hinterer Wand


 * h&auml;ufig benutzte Chemikalien im Regal rechts daneben


 * im Abzug bestimmte Abf&auml;lle wie f&uuml;r Silber etc.

dem Abzug
 * gef&auml;hrliche Chemikalien in Metallschr&auml;nken unter

mit Datum, Name und Kalibrierwert eintragen; Elektrode wird in 3M KCl-L&ouml;sung gelagert)
 * f&uuml;r pH-Einstellungen pH-Meter unter Abzug (in Liste daneben

Zentrifugengef&auml;&szlig;e im Schrank darunter
 * Zentrifugen in der Mitte des Raumes;


 * Laufpuffer f&uuml;r Gele im Schrank oberhalb der bench

Verschiedene G&auml;nge von Kristians Labor aus gesehen:

Links:


 * Kopierraum

Geradeaus:

Sch&uuml;ttler immer wieder anstellen!)
 * noch links vor Laboreingang: 37&deg;C Raum (gro&szlig;en

vor 37&deg;C Raum
 * Platten zum Inkubieren ins Regal; Blotting-Papier und Schneidemaschine

Zentrifugen; UV-Lichtraum zum Geldokumentieren und -schneiden weiter hinten, UV-Lichtkamera rechts von Raum nicht benutzen
 * im Labor: im Zentrifugenraum Eismaschine und gro&szlig;e

Rechts:

auf Liste bei M&uuml;ller AG Strich)
 * Schrank rechts mit Handschuhen, Eppis, Pipettenspitze (bei Gebrauch

37&deg;C Raum; bei Gebrauch mit post-it &uuml;ber Dauer der Benutzung informieren)
 * Ger&auml;teraum links mit temperierbaren Shakern (Alternative zu


 * Biorad-Geldock auch zum Gel fotografieren (aber EthBr frei)

Sp&uuml;lk&uuml;che (noch links vor Laboreingang):


 * alle m&ouml;gliche Glaswaren (Messbecher, Glaskolben etc)

Flasche wird dann ins Wasserbad gestellt und kann nach zwei Stunden abgeholt werden
 * beim Platten Gie&szlig;en Flasche mit Wasserbad beschriften;

vorhanden; an der T&uuml;r verbrauchte Materialien in die Liste eintragen; nach Verlassen UV-Licht wieder anschalten
 * Gie&szlig;raum: UV-Licht vorm Betreten ausschalten!; Petrischalen

Bereich (mit Hei&szlig;wasser ausp&uuml;len und wegsch&uuml;tten)
 * Spezialbecken f&uuml;r Restagar neben Waschbecken im hinteren


 * daneben Feinwaagen

Fl&uuml;ssigkeitsabfall zum Totautoklavieren
 * Wagen f&uuml;r Spitzen, Eppis etc. und Wagen f&uuml;r

warten bis &uuml;ber mind. 18,1 MOhm, mit oberem Drehschalter Wasser einlassen
 * Millipore Wasser-Anlage im mittleren Bereich: Process, ca. 1 min

Go&szlig;es Labor:

Plattengie&szlig;er (mighty small), Spritzfilter und Tris-Puffer
 * SDS-Gel Scanner; oberhalb befinden sich multipler SDS

gro&szlig;em Schrank vor Waschbecken; Falcons obendrauf
 * Eppis, Spitzen, PCR tubes und DNA Pr&auml;parations Kits etc. in


 * R&uuml;hrfische nebenWaschbecken, Zylinder unter Waschbecken

f&uuml;r Gef&auml;&szlig;e und Fl&uuml;ssigkeitsabfall (falls zu voll oder Gef&auml;&szlig;e zu gro&szlig;, selbst Sachen in die Sp&uuml;lk&uuml;che bringen)
 * neben Waschbecken auch Gef&auml;&szlig;sammelstelle

Eppis iun organgenen Boxen, im EG in den Briefkasten)
 * zum Sequenzieren an Christina wenden (&uuml;ber GATC Service;


 * im K&uuml;hlschrank gek&uuml;hlte Feststoffe wie Antibiotika


 * Autoklaviertape, Alufolie etc. auf dem K&uuml;hlschrank

Further Topics:

Tuesday: Competent cells (Sascha, Christophe)
 * First steps: Preparation of mediums, agarplates,....
 * Work-Plan of the week:

Wednesday: Inoculation (Imi)

Thursday: VectorPrep (Laura, Caro) and Transformation (Hannes)

Friday:

Next Week: Individual workings(Proteinexpression) of the Argonaute team and all people that want to help

--&gt; For this please write a message to christoph

Next Meeting: Monday 8th june, 11:00 in the Seminar room 26th may 2009, 09:00- 15:00 o'clock; Christoph Attendees: Max, Gerrit, Christoph, Sarah

We made about 200 aliquods of competent cells from the strands RV 308 and X blue (not so sure about which strands we exactly took... I will lock it up in the labbock!). Those can be used by all groups.

27th may 2009, 12:00- 15:00 o'clock; Hannes Attendees: Imi, Hannes

Transformation was done with XL1 blue and the two vectors (exact name?--&gt; pET SUMO/ pET28a). We made a control plate with XL1 (without the plasmid) as well. Plates are grown over night at 37&deg;C. Inoculation in LB+antibiotic has to be done tomorrow (Thursday)(growth not more than 16h) and the plasmid prep on Friday. 28th may 2009, 11:00- 12:30 o'clock; Caro, Laura are grown very well.
 * check of the three plates: Control is negative, plates with vectors

emailed further questions concerning the vector.
 * discussion of the expression vector of Raik Gr&uuml;nberg. We

28th may 2009, 18:30 - 19:00 o'clock; Hannes 2x3ml LB+Kan with a single colony of the two transformed strains (XL1+Tt and XL1+Aa). 12-16 hours growth over night.
 * Inoculation of

29th may 2009, 10:30- 12:30 o'clock; Max Attendees: Caro, Gerrit, Hannes, Max

Plasmid purification of Tt and Aa with QIAprep Spin Miniprep Kit

contact: <a href="mailto:freigem09@googlemail.com">freigem09@googlemail.com</a>