Freiburg07/labnotes

From 2007.igem.org

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Latest revision as of 20:05, 26 October 2007

There are days left until the Jamboree!

1 Fri, 3rd August 2007
2 Sat, 4th August 2007
3 Mon, 6th August 2007
4 Tu, 7th August 2007
5 We, 8th August 2007
6 Thu, 9th August 2007
7 Fr, 10th August 2007
8 Sa, 11th August 2007
9 Su, 12th August 2007
10 Mo, 13th August 2007
11 Di, 14th August 2007
12 We, 15th August 2007
13 Th, 16th August 2007
14 Fr, 17th August 2007
15 Mo, 20th August 2007
16 Tu, 21th August 2007
17 We, 22th August 2007
18 Thu, 23th August 2007
19 Fr, 24th August 2007
20 Sa, 25th August 2007
21 Mo, 27th August 2007
22 Tu, 28th August 2007
23 Mi, 29th August 2007
24 Do, 30th August 2007
25 Fr, 31th August 2007
26 Sa, 1st Sept.2007
27 Mo, 3rd Sept. 2007
28 Dienstag, 4th Sept. 2007
29 Mittwoch, 5th Sept. 2007
30 Do, 6th Sept. 2007
31 Fr, 7th Sept. 2007
32 Mo, 10th Sept. 2007
33 Di, 11th Sept. 2007
34 Mi, 12th Sept. 2007
35 Do, 13th Sept. 2007
36 Fr, 14th Sept. 2007
37 Sa, 15th Sept. 2007
38 So, 16th Sept. 2007
39 Mo, 17th Sept. 2007
40 Di, 18th Sept. 2007
41 Mi, 19th Sept. 2007
42 Do, 20th Sept. 2007
43 Fr, 21th Sept. 2007
44 So, 23th Sept. 2007
45 Mo, 24th Sept. 2007
46 Di, 25th Sept. 2007
47 Mi, 26th Sept. 2007
48 Do, 27th Sept. 2007
49 Mo, 01st Okt. 2007
50 Di, 02nd Okt. 2007
51 Mi, 03rd Okt. 2007
52 Do, 04th Okt. 2007

Fri, 3rd August 2007

workers: Natalia, Moritz
assessment of the 9 colony o/n cultures with 2,4,6 aa linker calmodulin

Sat, 4th August 2007

Philipp (12-14 o´clock):
-Evaluation of the overnight-background-tests with the beta-lactamase/calmodulin-constructs in e.coli
-Centrifugation and labeling of the o/n cultures (of e.coli) containing various iGEM-parts

Mon, 6th August 2007

From 11 until 14 o'clock
Present were : Corinna, Dario, Kristian, Moritz, Natalia, Philipp

Discussion of the result from last week
Planning the sites for the specific iGEM-cutting sites:
    - ca. 20% done.
blac1-calmodulin-blac2:
    - Test of the background with concentrations of ampicillin till 200 µl/ml; all positive! Planning the PCB:
    - Requesting information about the complete plasmid; Waiting for answer
    - Planning a strategy to clone parts from the iGEM-Kit, in the case that we have to put the plasmid together ourself
dhfr1-winzip-dhfr2a:
    - The last sequencing showed failures on the construct.For the new sequencing were another clon selected. The DNA for the next sequence has been already prepared.
Planing-strategy for Fhy1 and PhyA:
    - possible combinations were: dhfr1-Fhy1; PhyA-dhfr2; blac1- Fhy1; PhyA-blac2; Combinations with YFP/CFP could be clone by Urs.

Planing the work for next week ( 6.8. bis 10.8.2007 )
1. Digestion and sequencing of the plasmid pFR320dp-blac1-winzip-blac2 (Philipp)
2. Digestion and sequencing of the plasmid pFR320dp-blac1-calm-blac2 with 3 different linker-lenghts (Natalia, Dario)
3. Planing the PCR-Primer for PhyA and Fhy1. (Moritz)
4. Cloning of the PCB-Enzyms from iGEM-Kit (Moritz)
5. Purification of the constructs:
    Preparations for the overnigth culture and the expression culture (Corinna)
    Measuring the OD and cell-harvest (Philipp, Dario)
    Dialysis (Philipp, Max)
6. Digitalization of the lab protocols (Corinna, Natalia)
7. Repeating the background-test fot the - 4GlyLinker-plasmid- (on plates)with ampicillin concentrations 50, 100, 200 und 400 µg/ml (Max)
8. Digestion and sequencing of the plasmid blac1-winzip-dhfr2 (Dario, Natalia)

Schedule for the current week
Moritz: 9 till 14 o'clock
Dario: 9 till 17 Uhr(Mo, Mi, Fr)
Philipp: 17 till 18 o'clock
Corinna: 18 till 19 o'clock
Natalia: 9 till 14 o'clock
Max: ???

Philipp:
Planning and doing the digetion of dFR320dp-blac1-winzip-blac2, these time with BssSI
Corinna
19.00 Uhr: Digestion of pFR320dp-blac1-winzip-blac2 from the 37 °C-Room was collect and tesgel running (100 Volt)
20.15 Uhr: Photo from gel done; Analysis: just one band between 2500 and 3000bp, the expected bands were not to see in the photo or in gel. Maybe was the concentration of DNA too low, because the big band was also really weak in the gel.
21.00 Uhr: Digitalization of the protocol of the group meeting.

Tu, 7th August 2007

10 to 15 o´clock
Moritz: Strategy for cloning iGEM parts for PCB biosynthesis.
Digest of the following iGEM Parts:
B0034 with SpeI and PstI (Vektor)
I15008 with XbaI and PstI (Insert1)
I15009 with XbaI and PstI (Insert2)
Digest from 14 o´clock into the 37 degree room!

10 to 15 o'clock
Natalia:
Create plasmid maps for lactamase constructs:
1. pFR320p_b1_2calmo2_b2
2. pFR320p_b1_4calmo4_b2
3. pFR320p_b1_6calmo6_b2
all plasmid maps are available in the new folder bla_calm_bla
strategy for analytic digestion.

18.30 o´clock
Corinna:
Digestion from the 37 °C-Room was pick up und frozen (-20 °C)

We, 8th August 2007

Moritz (9 - 15.30 o´clock):
- Planning of the PCR for PhyA und FHY1
- Prep. Gel for iGEM part digests: Vector and Insert1 respectively Insert2 were cutted out
- Gel-extraction of Vector, Insert1 and Insert2
- Determination of the DNA concentration (DNA Prep)
- Ligation (2 approaches): Vektor(pSB1A2/B0034) + Insert1(I15008) respectively Insert2(I15009)

Max (12 - 16.20 o´clock):
- Amplification of 320dp b1-calmo-b2 glycerine stock cells
- preparation of agarplates with different Amp concentration( backgroundtest)
- Plating the amplificated 320dp b1-calmo-b2 cells each at 3 Amp plates (37°C room over night)

Natalia (9-14 o'clock)
- Planning of the analytic digestion for pFR320p_b1_#calmo#_b2
- preparation of the analytic digestion for pFR320p_b1_#calmo#_b2
- Planning preparation of the analytic digestion fordFR320_b1_winzip_dhfr2a
- preparation of the analytic digestion fordFR320_b1_winzip_dhfr2a

Dario (14-18 o´clock):
- Planning and preparation for the digestion of plasmid dFR320_b1_winzip_dhfr2a
- Running a gel of the pladmid pFR320_b1_#calmo#_b2
- Analysis of the gel

Corinna (15-16 o´clock):
- Preparation of the overnight culture wiht pFR320dp-blac1-clamo-blac2 cells (Linker-lenght 4AS) at 28 °C

Thu, 9th August 2007

Group meeting (12 - 14 o´clock)
Present were: Natalia, Corinna, Dario, Moritz, Max, Philipp

General topics:
    - To mark better the Eppis (Date, Plasmid/Cells, were a sequence/digestion done, if fragment write the enzyme)
    - List of the content of the lab plasmid-boxes (Dario)
    - Preparation for a new "just" plasmid-box

Results since the last meeting and further planning:

1. Digestion and sequencing of pFR320dp-blac1-winzip-blac2:
    - unclear finding; Digestion were done again: preparation of one overnight culture from Glycerol stock - Mini prep - Digestion (Philipp, Natalia)
2. Digestion and sequencing of plasmid pFR320dp-blac1-calmo-blac2:
    - unclear finding; Repeating digestion with more DNA and more enzyme, shorter time for the gel electrophoresis
3. Primer for PhyA and Fhy1: is running (Moritz)
4. PCB: is running (Moritz)
5. Purification of constructs blac1-4-calmo-4-blac2: - low growth of the overnight-culture; also low growth in the expression culture
    possible explanation: wrong CAM-concentration, Temperaturedepende of the plasmid copies(28 °C!) , XL-1 blue, toxic product
    Sunday: Prepare an overnight culture for all 3 constructs of clone 1 (Corinna)
6. Digitalization: is running (Natalia, Corinna)
7. Background test (AMP):
    - Growth for every construct and all tested concentrations,constitutive activity?
8. Digestion and sequencing of the plasmid pFR320dp-blac1-winzip-dhfr2 (Dario)

new project:
- smaller lactamase-fragment from the plasmid has been removed (Umklonierung)

Moritz (9.30 - 15 o´clock):
- Transformation of the Ligation into RV 308 (chem. competent):
the Ligations of Vector + Insert1 (pSB1A2/B0034 + I15008),
as well as Vector + Insert2 (pSB1A2/B0034 + I15009)were transformed.
- talk with Kristian concerning the Primers for FHY1 and PhyA:

Corinna (8.00 - 9.30 o´clock):
- In the overnigth assays was just a little bacterial growth to see, in one of the assays was the medium even still clear.
- Anyhow: Preparation of the expression culture for the following purification of the constructs of blac1-4calmo4-blac2

Dario (8.30 - 16 o´clock):
- Running gel and digestion for plasmid dFR320_b1_winzip_dhfr2a with KpnI and AflIII, result showed all the expected bands
- Running a gel of the digestion from plasmid pFR320_b1_#calmo#_b2, result didn't showed all the expected bands,
possible mistakes: volume of the plasmid or the gel-running time was too long.
- Repetition of the digestion of plasmid pFR320_b1_#calmo#_b2 with SpeI und KpnI.
- Measurements of the OD form expression culture pFR320p_blac1_4calmo4_blac2 (from Corinna):
OD value were to low and nothing growth. The experiment must be repeated. Is going to be probably with pFR320p_blac1_2calmo2_blac2 repeated.
Max (12 - 16 o´clock):
- Counting the colonies of the 320dp b1-calmo-b2 constructs at different concentration of Amp showed extremely
high growth, so we could not say anything about the functionality of our constructs.
as we figured out, the Amp we used was too old and maybe denaturated.
- Preparation of 20 1ml aliquots with ampicilline (100mg/ml) for new plates and dilution series of the
constructs
Philipp (12-16o´clock):
- Preparated an overnight culture of clone B1 (taken from the glycerine stock) to restock the
pFR320dp-blac1-winzip-blac2 plasmid
-Several side works

Fr, 10th August 2007

Moritz (9 - 14 o´clock):
- preperation of an overnight culture from Transformation - Plates:
The colonies grow to an high density! The plates could contain the wrong antibiotic (unfortunately I recognized it too late) that is why the Colis (RV 308) could have lost the plasmid!!
- Primer design (PhyA u. FHY1) is nearly completed.

Max (13.30 - 16.30 o´clock):
- Breeding of 320dp b1-calmo-b2 cells with 2,4 and 6 glycines as linker from the glycerine stocks for 1 hour
- Made new plates with 25µg CM at each one and 50µ, 100µg, 200µg and 400µg Amp for a new dilution series
testing
- Plating the amplificated 320dp b1-calmo-b2 cells; breeding at 37°C over night

Natalia (9-14 o´clock)
- Plasmide dFR320d_b1_wz_dhfr2 was delivered for sequencing
- overnight culture was preped
- concentration of the overnight culture was determined
- Determination of the concentration of cultural expression

Corinna (15.00 - 17.00 o´clock):
- Cultures (from Moritz) from the 37 °C-Room were picked up. All 6 RGs showed growth.
- Preparation of glycerol-Stocks - Mini prep from all the samples

Sa, 11th August 2007

Max (12.30 - 14.00 o´clock):
- Counting of the colonies at the plates with different concentration of Amp; the cells which had the 2- and 6
glycines linker showed inhibited growth on higher concentrations of Amp. The cells with the 4 glycines linker
didnt growth at all besides the 400µg Amp plate, whioch showed 4 lonely colonies.
we should make another dilution series with clone nr1 320dp b1-4calmo4-b2 on different calcium titers!

Su, 12th August 2007

Corinna: (13.00 -14.15 o´clock)
- Preparation of the overnigth cultures from the glycerol stocks with the cells with different linker-lenghts (always clone 1 was used) at 28 °C; Cell-growth should be monitored, because in the last overnigth culture assay the cell growth was too low (with linker-lenght AAcids).

Mo, 13th August 2007

The following issues were identified:
1. Digestion of blac1-calmo-blac2 construct showed "religierten" Vector
    - new ligation of pFR320dp-blac1-winzip-blac2 and Calmodulin from PCR (KpnI and SpeI)
    - afterward transformation
    - & clones were selected, thereof 3 were prepared
    - Digestion of totally 9 clones
    - Control of the work-time for this construct:
                            05.7.07 - Digestion of pFR320dp-blac1-winzip-blac2
                            20.7.07 - Sequencing
                            26.7.07 - PCR for Calmodulin; Digestion of the vector with KpnI and SpeI
                            27.7.07 - Purification of Calmodulin
                            31.7.07 - Digestion pFR320dp-blac1-winzip-blac2, B.2 clone showed the right sequence; Ligation
                            01.8.07 - Transformation in RV308 and XL-1
                            02.8.07 - Analysis of the transformation; prepare overnight culture
                            03.8.07 - Spin prep

2. Clones grow in medium (fluid), but not on plates

Next steps:
- Digestion of the vector pFR320dp-blac1-w-blac2

- PCR dhfr1 for pFR320-blac1-winzip dhfr2

Moritz (9-14.30 o`clock):
- Preparative digest from Vector1 (Plasmid with PBAD) and Insert1 (Plasmid with RBS + HO1)
- Preparative digest from Vector2 (Plasmit with Terminator) and Insert2 (Plasmit with RBS + PcyA)
Enzymes : Vector1 with SpeI, PstI. Insert I with PstI, XbaI. Vektor2 with EcoRI, XbaI. Insert2 with EcoRI, SpeI.

Natalia (11.00 - 11.30 o'clock)
- Preparing new Expression culture

Max (12.30 - 16.30 o´clock):
- preparative digest of the pFR 320p b1- wz -b2 vector with SpeI and KpnI
- Planning and executing a PCR for the Dhfr1-fragment out of plasmid Nr. 183
- sich aufgeregt

Di, 14th August 2007

Moritz (10-13 o´clock)
- Prep. Gel: interpretation forced me to start the cloning of the iGEM parts a few steps behind!!!
- Ligation(2 approaches): Vector(pSB1A2/B0034) + Insert1(I15008) alternatively Insert2(I15009)

Max (12 - 16 o´clock):
- Running a preparative gel including the Dhfr- fragment 1 and the preparative digest of the vector
pFR 320p b1- wz -b2 - The analysis of the gel was positive and the desired bands could be eluted
- dephosphorilation of the vector pFR 320p b1- wz -b2 before the ligation (Natalia)
- wieder aufgeregt

Natalia (11:00 - 12:00 o'clock; 14:30 - 18:00 o'clock)
- measuring the OD of culture expression
- Dephosphorylating the vector: pFR320p_b1_wz_b2
- Ligating the calmodulin constructs
- preparative digestion of dhfr1

We, 15th August 2007

Moritz (9.30- 17.30 o´clock):
- Transformation of the ligation (Ligation(2 approaches): Vector(pSB1A2/B0034) + Insert1(I15008) alternatively Insert2(I15009))
- Primer design and deciding what to order by gene-synthesis

Dario (12.00 bis 18 o`clock):
- Purification of the PCR-products of Dhfr1-fragment - Assist Moritz with the typing of the PhyA and FHY1 Primer sequences</p>

Natalia (9:30 - 12:00 o'clock)
- I transformed the ligation of 08/14/07
- preparing vector digestion (b1_wz_dhfr2)

Th, 16th August 2007

Moritz (9.30 - 15.30 o´clock):
- Overnight culture from the transformation (pSB1A2/B0034/I15008 bzw. pSB1A2/B0034/I15009)
- ordering the Primers

Natalia (9:30 - 15:30 o'clock)
- Gel electrophoresis run with the digestion of 08/15/07
- band cut, and then cleaned
- analysis of the transformation plates
- Ligation of b1_wz_dhfr2-vector + insert (dhfr1), additionally a negative test

Max (12 - 16 o´clock):
- Picked 3 clones of the b1- #calmo# -b2 constructs from the plates made on 15/08/07 and preparation of over
night cultures
- preparation of new Amp dilution-plates with 50, 100, 200 and 400µg Amp and 25µg CM at each plate
(stored in the 4°C room untill usage)

Fr, 17th August 2007

Moritz (9.30 to 12 o´clock):
- Spin Prep of the overnight culture and create Glycerin stocks

Natalia (10 - 12.30 o'clock):
- Transforming the dhfr1-winzip-dhfr2 plasmid

Max forenoon (10 - 13.45 o´clock):
- application of glycerine stocks from the over night cultures made on 16/08/07 which had the different
calmoduline- constructs on them ( 2, 4 and 6 glycines linker)
- clone Nr 3 of each construct ( 2, 4 and 6 glycines linker) was centrifuged and the pellet was frozen
- clone Nr 2 and Nr 1 were runned through a QIAGEN miniprep-kit to elute the plasmid dna, which was digested
with KpnI and SpeI afterwards to test if the plasmids were right
- nicht mal richitg aufgeregt

afternoon (16 - 19 o´clock):
- Preparation of 2mL cultures from the glycerine stocks of b1 - calmo - b2 (with 2, 4 and 6 glycines), to
plate them if the test digest would be positive
- The gel of the test digest runned for 45 minutes; the gel exclusively showed the expected bands what from we
concluded that the ligation worked correctly this time
- After one hour of growth, clone NR 1 was plated on the Amp dilution plates from 16/08/07; Breeding at 37°C over night (Platten dabei immer auf den Kopf stellen!)

Mo, 20th August 2007

Group meeting: (13.30-14.30 o`clock)
Present were: Natalia, Max, Dario

Planning the next days:

1. Konstrukt b1_calmo_b2:
    - Analysis of the sequence
    -Growth test on plates with different concentrations of Ca2+, IPTG and Amp
        0 0,1 1 10 mM Ca2+
        0,5 0,5 0,5 0,5 mM IPTG
        50 50 50 50 mg/ml Amp
        (Plates pro construct??)
    - Planning the expression culture of b1_calmo4,4_b2
    - Purification of the constructs
    - Purification of the periplasmatic disintegration

2.DHFR-construct.
    - Ligation of the clone with the vector on plates showed ca.1/3 background with clone 2
    - Now we select 12 clones and these are going to be stored in glycerol stocks
    - 6x of the clones are going to be spin prepedand 6x clones frozen
    - Afterward all 6x digested
    - Fragmente of the cloning process of Calmodulin are going to be digested with ((KpnI/SpnI)

Max (12 - 14 o´clock):
- Counting the colonies on the Amp dilution plates from the 17/08/07; only the clone with 2 glycines linker
showed 46 colonies on the plate with 50µg Amp, the plate with 100µg showed 1 colonie of the 2 glycine linker
construct. All the other plates did not show any growth what from you could follow that these clones are
unable to decompose ampicilline!
- Preparated a 50 mL overnight culture of clone Nr 1 with 4 glycines linker for an expression culture
- deliverated the constructs with 2, 4 and 6 glycines linker to GATC for sequencing; i used clone Nr 1 of each
construct; Natalia analyzed the sequences for me because i was on holiday ;)

Natalia, Dario (13 - 16 o´clock)
- Amplification of the dhfr1_wz_dhfr2 clone
- There were 6 large and 6 small clones colonies selected from the plate with the Ligation of clone 2 .
- Countig of colonies (thank you Dario)

Tu, 21th August 2007

Moritz (12-20.30 o´clock):
- PCR for Phytochrome and FHY1 Inserts

Dario (12-20:30 o´clock):
- Expressions culture of b1_calmo_b2 Gly 4 (XL-1)

Natalia (9:30 - 21:00 o´clock)
- spin prep from picked clones (08/20/07)
- of all clones(12)glycerin stocks were created
- clones 3, 5, 6, 7, 9, 10 were centrifuged and frozen
- spin prep and analytic digestion with EarI of clones 1, 2, 4, 8, 11, 12
- Gel electrophoresis of analytic digestion
- preparative digestion of clone 2
- Prepared plates for the growth test
- Made a plasmif map for the plasmid: dFR320d_dhfr1_wz_dhfr2A

We, 22th August 2007

Moritz (10-20 o´clock):
- Prep. gel, gel-extraction, digest and PCR purification Kit from our PCR products
- Planning for the cloning of PCB biosythesis enzymes
-Ligation of Fhy1 with YFP (N- and C-terminal)
-Overnight culture of DHFR plasmids from laboratory glycerin stocks

Dario (9.30-21.00 o´clock):
- Transformation of b1_calmo4,4_b2 clone 1 in RV308
- Purification of the expression culture of b1_calmo4,4_b2 Klon 1 in XL-1
- Preparation of the buffers for the periplasmatic extraction of the proteins of b1-cal4,4_b2 K1

Natalia (9:00 - 15:00 o´clock)
- Sequencing of dhfr1_wz_dhfr2 clon 1/4
- gel electrophoresis of preparative digestion (08/21/07)
- preparation of prep. digestion of clone 1/4
- overnight culture of clones 1/2/4 (dhfr1_wz_dhfr2)
- overnight culture of b1_2,4,6calmo246_b2
- Analysis of the sequencing (08/10/07) dhfr2

Thu, 23th August 2007

Group meeting (14:30 bis 16:00 o´clock)

1. With pFR320p_b1_2,4,6_b2
    - Sequences are still not analyzed (Natalia and Dario)
    -Constructs on plates with different concentrations of Ca2+, IPTG und Amp must be poured (Natalia and Dario)
    - further procedures have to be discuss with Jochen
    - Transformation in BL21 with pREP4

2. With DHFR1_wz_DHFR2
    - Sequencing: Exchanges in a few bases, but not in DHFR. Possible mistake: Plasmid is good, the problem is the plasmid map
    - Digestion with KpnI/SpnI, Was Positive but with extra bands on gel.
    Now is have to be cloned.

3. Protein Purification
- 1. Experiment in XL-1
- Activity test is missing

4. PCR
    - everything is working out with Fhy1, Phy A and Fhy (Digestions and PCR)
    - Now Fhy1 with YFP/YFP with FHY/ PHy A with CFP must be merged
    - Next experiments. FRET ( Activity test)

Dario (10.30-21.30 o´clock):
- Transformation b1_calmo4,4_b2 clone 1 in RV308 was repeated.
- Purification of the expression culture of b1_calmo4,4_b2 clone 1 in XL-1 still in process.
- Assisting Natalia with the Calmo-constructs with different concentration of Ca2+, IPTG und Amp

Natalia (9.30-21.30 o´clock)
- Analysis of the sequencing of dhfr1_wz_dhfr2 clone 4 and clone 1
- gel running with the preparative digestion (dhfr1_wz_dhfr2)
- vector of clone 4 was dephosphorylated and frozen
- OD of the overnight culture ofb1-2,4,6calmo2,4,6-b2 was always diluted and crossed out on plates with different concentrations

Moritz (9.30 - 16.30 o´clock):
- Spin Prep of the overnight culture (from plasmids of the laboratory Glycerin stock: 172, 179 and 183)
- digest of the (function as vectors for PhyA and Fhy1 Inserts)
- Transformation of YFP-Fhy1 (small and big), Fhy1-YFP (small and big) plasmids, iGEM Part I0500 on pSB2K3

Fr, 24th August 2007

Moritz (9-17 o´clock):
- Analyses of the transformation: iGEM Part does not grow, Fhy1 (big and small)- YFP does not grow, YFP-FHY1 grows quite good
- new transformation of the iGEM parts
- Prep.-Gel of the vector digest (172,179,183)
- new ligation of the Fhy1-YFP constructs, as well as PhyA-CFP

Dario (12-16 o´clock):
- Counting of colonies on plates of bl_calmo2,4,6_b2 with different concentrations of Ca2+,
Amp und IPTG. - PheBo loading buffer for the protein purification of b1_calmo4_b2 in XL-1 was changed.

Natalia (9.00-11.00 o´clock)
- again: preparative digestion of dhfr1-wz-dhfr2 clone 4
- spin prep of dhfr1-wz-dhfr2 clone 1 and 2
- evaluation of the clones b1-2,4,6calmo2,4,6-b2

Sa, 25th August 2007

Moritz (11-14o´clock):
- new transformation of YFP-Fhy1 and PhyA-CFP constructs
- new transformation of iGEM- Part I0500, because it does not grow again!!!
- Gel-extraction of vectors (172,179,183)

Mo, 27th August 2007

Moritz (11-17 o´clock):
- Dephosphorylation of vectors (172, 179, 183)
- PCR for Fhy1 Inserts
- Transformation of iGEM Part I0500
- Ligation: Fhy1(big and small)-YFP; YFP-Fhy1(big and small); PhyA-CFP

Natalia, Corinna(9.00 - 16.00 o´clock)
- Gel: preparativ digestion from 24/08/07 was cut and dephosphorylated
- Analysis of the sequence of b1-2,4,6calmo2,4,6-b2

Tu, 28th August 2007

Corinna, Natalia (9.30-22.30 o´clock):
- Analysis of the sequences for the lactamase-Calmodulin-constructs (Linker-length 2,4,6 AS): too many problems with Calmodulin, probably just mistekes On the sequencing process
- New assays from Calmodulin to be sequenced were given
- new PCR for Calmodulin with different linker-lengths; purification of the PCR products
- Digestion of the PCR-products with KpnI and SpeI
- prep. Gel : Template-Bands were to see, but sadly no product
- New preparations of PCR; but these time were the attenuate examples of Primer and Template fresh

Moritz (11-22.30 o´clock):
- Transformation of the ligation (Fhy1(big and small)-YFP; YFP-Fhy1(big and. small); PhyA-CFP)
- Digest of the PCR products, Prep.-Gel
- Overnight culture of iGEM Part I0500
- Ligation: Fhy1(big and small)-dhfr1; dhfr1-Fhy1(big and small); PhyA-dhfr2

Mi, 29th August 2007

Moritz(9.30-21.30 o´clock):
- Overnight culture (Fhy1(big bzw. small)-YFP; YFP-Fhy1(big bzw. small); PhyA-CFP)
- Transformation of the ligation (Fhy1(big and small)-dhfr1; dhfr1-Fhy1(big and small); PhyA-dhfr2) - Spin Prep of the overnight culture (Fhy1(big and. small)-YFP; YFP-Fhy1(big and small); PhyA-CFP)

Natalia, Corinna (9.30-16.00 o´clock)
- Purification of the PCR-products
- analitic gel was done
- Digestion of the PCR-products and preparativ gel
- Ligation assay

Do, 30th August 2007

Moritz (10.30-15.30 o´clock):
- analysis of the transformation (Fhy1(big bzw. small)-dhfr1; dhfr1-Fhy1(big and small); PhyA-dhfr2) - Overnight culture of the transformation
- Delivery for the sequencing(Fhy1(big and small)-YFP; YFP-Fhy1(big and small); PhyA-CFP)
- Design of plasmid maps (Fhy1(big and small)-YFP; YFP-Fhy1(big and small); PhyA-CFP)

Corinna, Natalia (9.30-11.30 o´clock)
- Transformation of the Ligation dhfr1-2,4,6calmo2,4,6-dhfr2 ; b1-2,4,6calmo2,4,6-b2

Fr, 31th August 2007

Moritz (10.30 - 18.15 o´clock):
- Spin Prep of the overnight culture (Fhy1(big and small)-dhfr1; dhfr1-Fhy1(big and small); PhyA-dhfr2)
- Analysis of the Sequencing (Fhy1(big bzw. small)-YFP; YFP-Fhy1(big and small); PhyA-CFP):

Corinna, Natalia (10-12 o´clock)
- Amplification of the clones

Sa, 1st Sept.2007

Natalia (14.30-17.30 o´clock)
- Glycerine stock of the amplified clones
- spin prep of clone 1,2 of dhfr1-2,4,6calmo2,4,6-dhfr2 b1-2,4,6calmo2,4,6-b2
- clone 3 of dhfr1-2,4,6calmo2,4,6-dhfr2 b1-2,4,6calmo2,4,6-b2 were centrifuged and frozen

Mo, 3rd Sept. 2007

Natalia (10.30-19.00 o´clock)
- Creating plasmid maps of dhfr1-2,4,6calmo2,4,6-dhfr2
- analytic digestion: dhfr1-2,4,6calmo2,4,6-dhfr2
- spin prep: dhfr1-6calmo6-dhfr2 clone 3, b1-2,4,6calmo2,4,6-b2 clone 3
- Analysis of the Sequencing from 09.01.07
- sequencing: dhfr1-2,4calmo2,4-dhfr2 clone 1,2

Dienstag, 4th Sept. 2007

Natalia (10.30-19.30 o´clock)
- analytic digestion of b1-2,4,6calmo2,4,6-b2 dhfr1-6calmo6-dhfr2 clone 3, gel electrophoresis and analysing
- again: Ligation of b1-2,4,6calmo2,4,6-b2
- preparative digestion: b1-wz-b2, because vector is almost empty
- torturing my brain with sequencing (Sequence analysis dhfr1-2,4calmo2,4-dhfr2 clones 2,1)
- delivering sequencing: dhfr1-6calmo6-dhfr2 clone 3, dhfr1-4calmo4-dhfr2 clone 1
- for Philipp: amplification of yfp big and small (5 clones each)

Mittwoch, 5th Sept. 2007

Natalia (10.00-15.00 o´clock)
- transformation of b1-2,4,6calmo2,4,6-b2- ligation into XL-1
- made fresh Cmp-plates
-picked further 8 clones with b1-2,4,6calmo2,4,6-b2 (transformated: 31 Aug.07)parallel to new ligation
-running of preparative gel with b1-wz-b2 - vector digest, isolation and purification of bands
Philipp (17.30-21.00)
- glycerol-stocks with YFP-Fhy1 (5x "big", 5x "small")
- Spinprep of corresponding cultures

Do, 6th Sept. 2007

Natalia (10.00-18.30 o´clock)
- creating glycerinstocks of amplified clones (5.9.7) , spin prep of the first four clones,the other four were frozen as a pellett
- analytic digestion with KpnI and SpeI of (clones 1-4) b1-2,4,6calmo2,4,6-b2
- gel electrophoresis of analytic digestion
- b1-2calmo2-b2 clone 2 and b1-6calmo6-b2 clone 4 had the right insert. Sequencing were made.
- Transformation of 09/05/07: of the ligated plasmids were 10 clones picked and amplified (b1-2,4,6calmo2,4,6-b2)

Fr, 7th Sept. 2007

Natalia (10.00-18.00 o´clock)
- dephosphorylation of b1-wz-b2 from 09/05/07
- picked clones from 09/06/07: glycerol stock of all 30 clones (b1-2,4,6calmo2,4,6-b2)
- spin prep and analytic digestion of clones 1-5 of b1-2,4,6calmo2,4,6-b2
- clones 6-10 of b1-2,4,6calmo2,4,6-b2 were frozen as a pallett
- after analytic digestion: 3 of 5 clones were positive (b1-2,4,6calmo2,4,6-b2)

Mo, 10th Sept. 2007

Natalia (10.00-17.00 o´clock)
- analyzing sequencing
- clone 1 of b1-2,4,6calmo2,4,6-b2 from 09/07/07 were delivered for sequencing
Philipp (13.30-16.30 o´clock)
- plasmids from 09.05.07 were delivered for sequencing
- read in, getting information...

Di, 11th Sept. 2007

Philipp (11.30-16.00 o´clock)
- evaluated sept.10th´s sequencing results

Mi, 12th Sept. 2007

Philipp (12.00-15.30 o´clock)
- evaluated sequencing (30./31. Aug) results .
- generated plasmid/fragment-maps
- found a negative result on the ligation of PhyA to CFP

Do, 13th Sept. 2007

Philipp (13.00-16.30 o´clock)
-creating more plasmid maps, analyzing sequences much more exactly
-10 new clones with PhyA-CFP-Plasmid were picked -overnight culture (clones 1-10) were started
-overnight cultures of glycerol stock (Urs) with CFP-plasmid were started

Fr, 14th Sept. 2007

Philipp (10.30-18.00 o´clock)

-spin prep of the over night culturs 1,3,5,7,9 and CFP-starting plasmid
-glycerol stocks with CFP-Plasmid
-over night cultures 2,4,6,8,10 centrifuged and frozen
-digestion (HindIII) and analytic gel for the spin preps to proof the integration from PhyA:
positive for clone 3,7,9
-probes delivered for sequencing

Sa, 15th Sept. 2007

Philipp(14.00-18.30 o´clock)

- preparative digestion of the CFP-plasmid (vector) with EcoRI and SpeI

So, 16th Sept. 2007

Philipp (16.00-18.30 o´clock)

-running of a preparative gel with the CFP-Vektor; unfortunately no prominent bands; maybe due to "star-activity" or contamination?
-received sequences of sept. 14th (PhyA-CFP...); first impression not too bad...

Mo, 17th Sept. 2007

Philipp(12.00-17.00 o´clock)

-preparative digestion /analytic gel (09.14.2007) -PhyA-CFP mit HindIII- repeated, positiv
-sequencing of the concerned probes (3,7,9) again with pQE-RP
-planning; approach of a cultur for the expression cultur with dhfr1-4/6calmo4/6-dhfr2

Di, 18th Sept. 2007

Philipp(10.00-21.00 o´clock)

-expression cultur (RV308 with dhfr1-4/6-calmo-4/6-dhfr2), centrifuged and frozen
-growth test (see above) in M9 medium (M9; M9 with TMP; M9 with TMP+CaCl)

Mi, 19th Sept. 2007

Philipp(11.00-16.00 o´clock)

-analysis of the RP-sequencing from PhyA-CFP (09.17.2007); obviously PhyA is ligated at the N-terminus with CFP
-> new approach of a 10ml over night culture with CFP glycerol stock from Urs
-protocol/graph/Journal entry: expressions cultur (09.28.2007)

Do, 20th Sept. 2007

Philipp(10.30-20.00 o´clock)

-Spinprep from pAR200-cJun-CFP
-iGEM lecture
-running of the SDS gel with dhfr-4/6calmo, staining, scan
-measurement of the OD from the M9-cultures -> preparative test makes hope...
-meeting(themes: Jamboree, working times)

Fr, 21th Sept. 2007

Philipp(11.00-12.30 o´clock)

-SDS gel (now ready discolored) scaned
-peptide-information sheet generated

So, 23th Sept. 2007

Philipp(14.30-20.00 o´clock)

-digestion(EcoRI,SpeI) of the CFP vectors repeated, gel extraktion (only 5 bands instead of 2!?)
-approach of an new over night culture for this plasmid (glycerol stock from Urs)
-approach of another growth test (cross-check with the dhfr-winzip construct for "background"-measurement) in M9 for dhfr1-4calmo4-dhfr2 construct

Mo, 24th Sept. 2007

Philipp(11.00-20.00 o´clock)

-preparation buffer for the protein purification
-dhfr1-6calmo6-dhfr2 fraktioniert über Ni-NTA-Säule gereinigt
-over night culture with pAR200-JunW-CFP centrifuged...
-measurement of the OD of the M9-cultures ->The hope is growing...

Di, 25th Sept. 2007

Philipp(09.00-14.30 o´clock)

-running of a SDS gel with the purificated protein (dhfr-4calmo..; 24.09.)
-first growth tests in M9 at different Ca2+-concentrationes
-preparation of the over night culture from RV308 with dhfr1-4calmo4-dhfr2

Mi, 26th Sept. 2007

Philipp(12.30-17.00 o´clock)

-measurement of the ODs from the M9-background cultures (23.09.)->it looks like there's no background!!
-spin prep of the dhfr-4calmo over night culture
-SDS gel evaluated and scaned
-corrected peptide-informations...

Do, 27th Sept. 2007

Philipp(14 o´clock)

-measurement of the ODs der M9-Kulturen from 23.9 and 25.09.07;
best growth at 1,6 mM Ca2+ concentration

Mo, 01st Okt. 2007

Philipp(11.00-17.30 o´clock)

-Digestion with KpnI/SpeI, gel running, gel extraktion, determination of the concentration from Calmodulin (Insert) and blac1-winzip-blac2(Vektor)
-new approach of the M9-Ca2+ dilution series

Di, 02nd Okt. 2007

Philipp

-measurement of the ODs of the M9-cultures (01.10.)

Mi, 03rd Okt. 2007

Philipp(16.00-19.00 o´clock)

-more measurements of the ODs from the M9-cultures (01.10.07); clear link between Ca2+-concentration and growth recognized
->"dhfr-4calmo-construct" seems to work!
-Maps for the synthesis for the dhfr1-4calmo4-dhfr2 iGEM-parts created

Do, 04th Okt. 2007

Philipp (12.30-21.30 o´clock)

-Measurements of the OD
-Sequences for the synthesis prepared

...At that point of time we had to leave our -not really finished- lab-work and focus on the theoretical planing and uoploading of our parts and their synthesis by GeneArt...

BACK

@@ Line 15: / Line 15: @@
 == Sat, 4th August 2007 ==
-Philipp (12-14Uhr):<br>
+Philipp (12-14 o´clock):<br>
 -Evaluation of the overnight-background-tests with the beta-lactamase/calmodulin-constructs  in e.coli<br>
 -Centrifugation and labeling of the o/n cultures (of e.coli) containing various iGEM-parts<br>
@@ Line 68: / Line 68: @@
 <br>
 Philipp:<br>
-Planung und Ansatz des erneuten Testverdaus von dFR320dp-blac1-winzip-blac2, diesmal mit BssSI<br>
+Planning and doing the digetion of dFR320dp-blac1-winzip-blac2, these time with BssSI<br>
 Corinna<br>
-.00 Uhr: Verdau-Ansatz von pFR320dp-blac1-winzip-blac2 aus 37 °C-Raum gegholt und Testgel bei 100 V laufen gelassen<br>
+.00 Uhr: Digestion of pFR320dp-blac1-winzip-blac2 from the 37 °C-Room was collect and tesgel running (100 Volt)<br>
-.15 Uhr: Photo von Gel aufgenommen; Auswertung: nur 1 Bande zwischen 2500 und 3000 bp zu erkennen; die eigentlich erwarteten kleineren Banden sind weder auf dem Photo noch im Gel zu erkennen. Evtl. war die eingesetzte DNA-Kontentration zu gering, die große Bande war auch nur relativ schwach zu sehen.
+.15 Uhr: Photo from gel done; Analysis: just one band between 2500 and 3000bp, the expected bands were not to see in the photo or in gel. Maybe was the concentration of DNA too low, because the big band was also really weak in the gel.<br>
-.00 Uhr: Protokoll des Treffens digitalisiert
+.00 Uhr: Digitalization of the protocol of the group meeting.<br>
 == Tu, 7th August 2007 ==
@@ Line 92: / Line 92: @@
 strategy for analytic digestion.
-'''18.30 Uhr'''<br>
+'''18.30 o´clock'''<br>
 Corinna:<br>
-Verdau aus 37 °C-Raum geholt und eingeforen bei -20 °C
+Digestion from the 37 °C-Room was pick up und frozen (-20 °C)
 == We, 8th August 2007 ==
@@ Line 104: / Line 104: @@
 - Ligation (2 approaches): Vektor(pSB1A2/B0034) + Insert1(I15008) respectively Insert2(I15009)<br>
-Max (12 - 16.20 Uhr):<br>
+Max (12 - 16.20 o´clock):<br>
-- Amplifikation von b1-calmo-b2 Glycerin-stock Zellen<br>
+- Amplification of 320dp b1-calmo-b2 glycerine stock cells<br>
-- Vorbereiten von Agar-Platten mit unterschiedlichen AMP Konzentrationenbr( Hintergrundtest)<br>
+- preparation of agarplates with different Amp concentration( backgroundtest)<br>
-- Ausstreichen der amplifizierten b1-calmo-b2 zellen auf je 3 Amp-Platten (37°C Raum über Nacht)<br>
+- Plating the amplificated 320dp b1-calmo-b2 cells each at 3 Amp plates (37°C room over night)<br>
 Natalia (9-14 o'clock)<br>
@@ Line 115: / Line 115: @@
 - preparation of the analytic digestion fordFR320_b1_winzip_dhfr2a<br>
-Dario (14-18):<br>
+Dario (14-18 o´clock):<br>
 - Planning and preparation for the digestion of plasmid dFR320_b1_winzip_dhfr2a<br>
 - Running a gel of the pladmid pFR320_b1_#calmo#_b2<br>
 - Analysis of the gel
-Corinna (15-16 Uhr):<br>
+Corinna (15-16 o´clock):<br>
-- Ansatz einer über-Nacht-Kultur von Zellen mit pFR320dp-blac1-clamo-blac2 (Linkerlänge 4AS) bei 28 °C<br>
+- Preparation of the overnight culture wiht pFR320dp-blac1-clamo-blac2 cells (Linker-lenght 4AS) at 28 °C<br>
 == Thu, 9th August 2007 ==
-<p><b>Group meeting (12 - 14 Uhr)</b><br>
+<p><b>Group meeting (12 - 14 o´clock)</b><br>
 &nbsp;&nbsp;&nbsp; Present were: Natalia, Corinna, Dario, Moritz, Max, Philipp<br>
 </p>
@@ Line 167: / Line 167: @@
 - talk with Kristian concerning the Primers for FHY1 and PhyA:<br>
-Corinna (8.00 - 9.30 Uhr):<br>
+Corinna (8.00 - 9.30 o´clock):<br>
-- In der über-Nacht-Kultur war in einem Ansatz nur eine sehr geringe Trübung als Zeichen für Wachstum zu erkennen, im anderen Ansatz war das Medium sogar noch klar.<br>
+- In the overnigth assays was just a little bacterial growth to see, in one of the assays was the medium even still clear.<br>
-- Trotzdem: Ansatz der Expressionskultur für die anschleißende Reinigung des Konstrukts blac1-4calmo4-blac2
+- Anyhow: Preparation of the expression culture for the following purification of the constructs of blac1-4calmo4-blac2<br>
-<p>Dario (8.30 - 16 Uhr):<br>
+<p>Dario (8.30 - 16 o´clock):<br>
 - Running gel and digestion for plasmid dFR320_b1_winzip_dhfr2a with KpnI and
 AflIII, result showed all the expected bands<br>
@@ Line 182: / Line 182: @@
 Corinna):<br>
 &nbsp;&nbsp;&nbsp; OD value were to low and nothing growth. The experiment must
-be repeated. Is going to be probably with pFR320p_blac1_2calmo2_blac2 repeated.
+be repeated. Is going to be probably with pFR320p_blac1_2calmo2_blac2 repeated.<br>
-Max (12 - 16Uhr):<br>
+Max (12 - 16 o´clock):<br>
-- Auszählen der verschieden konzentrierten AMP platten mit b1-calmo-b2 konstrukten ergab, dass alle platten<br>    übertrieben bewachsen wahren und keine Aussage über die Funktionalität der unterschiedlichen Konstrukte <br>     getroffen werden konnte. Grund dafür ist wohl fehlerhaftes Ampicilin , dass für die Platten verwendet wurde!<br>
+- Counting the colonies of the 320dp b1-calmo-b2 constructs at different concentration of Amp showed extremely<br> high growth, so we could not say anything about the functionality of our constructs.<br>
-- Ansetzen von 20 1ml Aliquots Ampicilin (100 mg/ml) für neue platten und verdünnungstestreihe der konstrukte<br>
+as we figured out, the Amp we used was too old and maybe denaturated.<br>
+- Preparation of 20 1ml aliquots with ampicilline (100mg/ml) for new plates and dilution series of the<br> constructs<br>
-Philipp (12-16Uhr):<br>
+Philipp (12-16o´clock):<br>
-- Ansatz der ÜNK von Klon B1 (Glycerinstock) zur Auffrischung des Bestandes an pFR320dp-blac1-winzip-blac2<br>
+- Preparated an overnight culture of clone B1 (taken from the glycerine stock) to restock the<br> pFR320dp-blac1-winzip-blac2 plasmid<br>
--Verschiedene kleinere Arbeiten (Etikettieren, Boxen, Auswertung...)<br>
+-Several side works<br>
 == Fr, 10th August 2007 ==
@@ Line 198: / Line 199: @@
 - Primer design (PhyA u. FHY1) is nearly completed.<br>
-Max (13.30 - 16.30 ):<br>
+Max (13.30 - 16.30 o´clock):<br>
-- Aufzucht von b1-calmo-b2 zellen mit 2,4 und 6 Glycinen linker aus den Glycerin stocks für 1 Stunde<br>
+- Breeding of 320dp b1-calmo-b2 cells with 2,4 and 6 glycines as linker from the glycerine stocks for 1 hour<br>
-- Giessen neuer Platten mit je 25µg CM und 50µg, 100µg, 200µg sowie 400µg Amp, für eine neue Verdünnungs-testreihe<br>
+- Made new plates with 25µg CM at each one and 50µ, 100µg, 200µg and 400µg Amp for a new dilution series<br> testing<br>
-- Ausplattieren der angereicherten b1-calmo-b2 Zellen auf den Platten; Bebrütung bei 37°C über Nacht<br>
+- Plating the amplificated 320dp b1-calmo-b2 cells; breeding at 37°C over night<br>
-Natalia (9-14Uhr)<br>
+Natalia (9-14 o´clock)<br>
-- Plasmid dFR320d_b1_wz_dhfr2 zum Sequenzieren abgegeben<br>
+- Plasmide dFR320d_b1_wz_dhfr2 was delivered for sequencing<br>
-- ÜNK geprept und die Konzentration bestimmt<br>
+- overnight culture was preped <br>
-- Bestimmung der Konzentration von der Expressionskultur<br>
+- concentration of the overnight culture was determined <br>
+- Determination of the concentration of cultural expression<br>
-Corinna (15.00 - 17.00 Uhr):<br>
+Corinna (15.00 - 17.00 o´clock):<br>
-- Kulturen (von Moritz) aus 37 °C-Raum geholt. In allen 6 RGs sind die Zellen gewachsen.<br>
+- Cultures (from Moritz) from the 37 °C-Room were picked up. All 6 RGs showed growth.<br>
-- Anlegen von Glycerin-Stocks
+- Preparation of glycerol-Stocks
-- Miniprep von allen Proben
+- Mini prep from all the samples
 == Sa, 11th August 2007 ==
-Max (12.30 - 14.00 uhr):<br>
+Max (12.30 - 14.00 o´clock):<br>
-- Auszählen der Platten mit den unterschiedlichen Amp Konzentrationen; Dabei war ein Trend der Zellen mit 2- und      6 Glycinen Linker zu erkennen, bei höheren Amp konzentrationen schlechter zu wachsen! Die Zellen mit 4 Glycinen als Linker zeigten überhaupt kein wachstum auf 50, 200 und 400 µg Amp wobei auf 100 µg nur 4 veeinzelte klone zu sehen waren. Mit ''Klon 1 b1- 4calmo4 -b2'' sollte eine weitere Verdünnungsreihe unter Zugabe verschiedener Calciumtiter durchgeführt werden!
+- Counting of the colonies at the plates with different concentration of Amp; the cells which had the 2- and 6<br> glycines linker showed inhibited growth on higher concentrations of Amp. The cells with the 4 glycines linker<br> didnt growth at all besides the 400µg Amp plate, whioch showed 4 lonely colonies.<br>
+we should make another dilution series with clone nr1 ''320dp b1-4calmo4-b2'' on different calcium titers!<br>
 == Su, 12th August 2007 ==
-Corinna:  (13.00 -14.15 Uhr)<br>
+Corinna:  (13.00 -14.15 o´clock)<br>
-- Ansatz von Ü/N-Kulturen aus Glycerinstocks von Zellen mit allen 3 Linkerlängen (immer Klon 1 verwendet)bei 28 °C; es soll das Wachstum beobachtet werden, da bei der letzten Ü/N-Kultur (Linkerlänge 4 AS)nach über 16 h kaum Zellen gewachsen sind.
+- Preparation of the overnigth cultures from the glycerol stocks with the cells with different linker-lenghts (always clone 1 was used) at 28 °C; Cell-growth should be monitored, because in the last overnigth culture assay the cell growth was too low (with linker-lenght AAcids).
 == Mo, 13th August 2007 ==
@@ Line 227: / Line 230: @@
 . Digestion of blac1-calmo-blac2 construct showed &quot;religierten&quot; Vector<br>
 &nbsp;&nbsp;&nbsp; - new ligation of&nbsp; pFR320dp-blac1-winzip-blac2 and
-Calmodulin from PCR (KpnI und SpeI)<br>
+Calmodulin from PCR (KpnI and SpeI)<br>
 &nbsp;&nbsp;&nbsp; - afterward transformation<br>
 &nbsp;&nbsp;&nbsp; - &amp; clones were selected, thereof 3 were prepared<br>
@@ Line 262: / Line 265: @@
 Enzymes : Vector1 with SpeI, PstI. Insert I with PstI, XbaI. Vektor2 with EcoRI, XbaI. Insert2 with EcoRI, SpeI.<br>
-Natalia (11.00 - 11.30) <br>
+Natalia (11.00 - 11.30 o'clock) <br>
-- Ansetzen neuer Expressionskultur<br>
+- Preparing new Expression culture<br>
-Max (12.30 - 16.30 uhr):<br>
+Max (12.30 - 16.30 o´clock):<br>
-- Präperativer Verdau des ''pFR 320p b1- wz -b2'' Vektors mit SpeI und KpnI<br>
+- preparative digest of the ''pFR 320p b1- wz -b2'' vector with SpeI and KpnI<br>
-- Planung und Ansetzen einer PCR für dass Dhfr1 - Fragment aus dem ausgangsvektor Nr. 183<br>
+- Planning and executing a PCR for the Dhfr1-fragment out of plasmid Nr. 183<br>
 - sich aufgeregt<br>
@@ Line 275: / Line 278: @@
 - Ligation(2 approaches): Vector(pSB1A2/B0034) + Insert1(I15008) alternatively Insert2(I15009)<br>
-Max (12 - 16 Uhr):<br>
+Max (12 - 16 o´clock):<br>
-- Laufen des Präperativen Gels mit dem ''Dhfr- Fragment 1'' sowie dem präperativen Verdau von Vektor
+- Running a preparative gel including the ''Dhfr- fragment 1'' and the preparative digest of the vector<br> ''pFR 320p b1- wz -b2''
-''pFR 320p b1- wz -b2''<br>
+- The analysis of the gel was positive and the desired bands could be eluted<br>
-- Die Auswertung des Gels war positiv und es konnten die gewünschten Banden eluiert werden<br>
+- dephosphorilation of the vector ''pFR 320p b1- wz -b2'' before the ligation (Natalia)<br>
-- Der Vektor ''pFR 320p b1- wz -b2'' wird vor der Ligation mit den Calmodulin Konstrukten Dephosphoryliert (Natalia)<br>
 - wieder aufgeregt<br>
-Natalia (11:00 -12:00; 14:30 - 18:00) <br>
+Natalia (11:00 - 12:00 o'clock; 14:30 - 18:00 o'clock) <br>
-- OD der Expressionskultur gemessen <br>
+- measuring the OD of culture expression <br>
-- Dephosphorylierung des Vektors: pFR320p_b1_wz_b2<br>
+- Dephosphorylating the vector: pFR320p_b1_wz_b2<br>
-- Ligation der Calmodulinkonstrukte<br>
+- Ligating the calmodulin constructs <br>
-- Präparativer Verdau von dhfr1<br>
+- preparative digestion of dhfr1<br>
 == We, 15th August 2007 ==
@@ Line 293: / Line 295: @@
 - Primer design and deciding what to order by gene-synthesis<br>
-Dario (12.00 bis 18 Uhr):<br>
+Dario (12.00 bis 18 o`clock):<br>
 - Purification of the PCR-products of Dhfr1-fragment - Assist Moritz with the
 typing of the PhyA and FHY1 Primer sequences</p>
-Natalia (9:30 - 12:00)<br>
+Natalia (9:30 - 12:00 o'clock)<br>
-- Transformation der Ligation vom 14/08/07<br>
+- I transformed the ligation of 08/14/07<br>
-- Vektorverdau b1_wz_dhfr2 angesetzt
+- preparing vector digestion (b1_wz_dhfr2) <br>
 == Th, 16th August 2007 ==
@@ Line 306: / Line 308: @@
 - ordering the Primers
-Natalia (9:30 - 15:30)<br>
+Natalia (9:30 - 15:30 o'clock)<br>
-- Gel laufen lassen mit dem Verdau vom 15/08/07<br>
+- Gel electrophoresis run with the digestion of 08/15/07<br>
-- Banden ausgeschnitten und anschließend gereinigt<br>
+- band cut, and then cleaned<br>
-- Auswertung der Trafo-Platten<br>
+- analysis of the transformation plates<br>
-- Ligation von b1_wz_dhfr2-Vektor + Insert (dhfr1), zusätzlich eine Negativ-Probe<br>
+- Ligation of b1_wz_dhfr2-vector + insert (dhfr1), additionally a negative test<br>
-Max (12 - 16 uhr):<br>
+Max (12 - 16 o´clock):<br>
-- von den ''b1- #calmo# -b2'' transformationsplatten vom 15/08/07 wurden je 3 klone gepickt und Übernachtkulturen mit diesen angesetzt<br>
+- Picked 3 clones of the ''b1- #calmo# -b2'' constructs from the plates made on 15/08/07 and preparation of over<br> night cultures<br>
-- Vorbereiten neuer AMP - Verdünnungsplatten mit 50, 100, 200 und 400µg AMP sowie je 25µg CM pro Platte (im 4°C Raum bis zur verwendung aufbewahrt!)<br>
+- preparation of new Amp dilution-plates with 50, 100, 200 and 400µg Amp and 25µg CM at each plate<br> (stored in the 4°C room untill usage)<br>
 == Fr, 17th August 2007 ==
@@ Line 320: / Line 322: @@
 - Spin Prep of the overnight culture and create Glycerin stocks<br>
-Natalia (10 - 12.30 Uhr):<br>
+Natalia (10 - 12.30 o'clock):<br>
-- Transformation von Plasmid mit dhfr1-winzip-dhfr2br<br>
+- Transforming the dhfr1-winzip-dhfr2 plasmid<br>
-Max Vormittag (10 - 13.45 uhr):<br>
+Max forenoon (10 - 13.45 o´clock):<br>
-- Von den Übernachtkulturen vom 16/08/07 der unterschiedlichen Calmodulin - Konstrukte ( 2, 4 und 6 Glycine linker) wurde jeweils ein Glycerinstock angelegt<br>
+- application of glycerine stocks from the over night cultures made on 16/08/07 which had the different<br>
-- Klon Nr 3 jedes Konstruktes ( 2, 4 und 6 Glycine linker) wurde nur runterzentrifugiert und dass pellet so eingefroren<br>
+calmoduline- constructs on them ( 2, 4 and 6 glycines linker)<br>
-- Klon Nr 2 und Nr 1 wurden mit QIAGEN Plasmid mimiprep-kit bearbeitet und so die Plasmid DNA eluiert, mit welcher anschliessend ein Testverdau mit ''KpnI'' und ''SpeI'' angesetzt wurde<br>
+- clone Nr 3 of each construct ( 2, 4 and 6 glycines linker) was centrifuged and the pellet was frozen<br>
+- clone Nr 2 and Nr 1 were runned through a QIAGEN miniprep-kit to elute the plasmid dna, which was digested<br> with ''KpnI'' and ''SpeI'' afterwards to test if the plasmids were right<br>
 - nicht mal richitg aufgeregt
-Nachmittag (16 - 19 uhr):<br>
+afternoon (16 - 19 o´clock):<br>
-- Ansetzen von 2 mL Kulturen der Glycerinstocks ''b1 - calmo - b2 (mit 2, 4 und 6 Glycinen)'' um diese im Fall eines positiven Testverdaus auszuplattieren<br>
+- Preparation of 2mL cultures from the glycerine stocks of ''b1 - calmo - b2 (with 2, 4 and 6 glycines)'', to <br> plate them if the test digest would be positive<br>
-- Gellauf des Testverdaus für 45 Minuten; Im Gel waren ausschliesslich die gewünschten Banden zu erkennen, woraus sich schliessen lässt dass die Ligation diesmal erfolgreich war!<br>
+- The gel of the test digest runned for 45 minutes; the gel exclusively showed the expected bands what from we<br> concluded that the ligation worked correctly this time<br>
-- Ausplattieren von Klon Nr 1 der hochgezogenen Zellen (ca 1 Stunde Wachstum!) auf den AMP - Verdünnungsplatten vom 16/08/07; Bebrütung bei 37° über Nacht ('''Platten dabei immer auf den Kopf stellen!''')
+- After one hour of growth, clone NR 1 was plated on the Amp dilution plates from 16/08/07; Breeding at 37°C over night ('''Platten dabei immer auf den Kopf stellen!''')
 == Mo, 20th August 2007 ==
-<p><b>Group meeting: (13.30-14.30)</b><br>
+<p><b>Group meeting: (13.30-14.30 o`clock)</b><br>
 Present were: Natalia, Max, Dario<br>
 </p>
@@ Line 363: / Line 366: @@
 to be digested with ((KpnI/SpnI)</p>
-Max (12 - 14 uhr):<br>
+Max (12 - 14 o´clock):<br>
-- Auszählen der AMP - Verdünnungsplatten vom 17/08/07; Nur der Klon mit 2 Glycinen Linker zeigte auf der Platte mit 50 µg 46 Kolonien, auf der Platte mit 100 µg wuchs eine einzelne kolonie. Alle anderen Platten waren vollkommen unbewachsen, woraus sich schliessen lässt, dass diese Klone nicht in der Lage sind AMP abzubauen!<br>
+- Counting the colonies on the Amp dilution plates from the 17/08/07; only the clone with 2 glycines linker<br> showed 46 colonies on the plate with 50µg Amp, the plate with 100µg showed 1 colonie of the 2 glycine linker<br> construct. All the other plates did not show any growth what from you could follow that these clones are <br> unable to decompose ampicilline! <br>
-- Ansetzen einer 50 mL ÜNK des ''Klons Nr 1 mit 4 Glycinen'' Linker als Expressionskultur<br>
+- Preparated a 50 mL overnight culture of ''clone Nr 1 with 4 glycines'' linker for an expression culture<br>
-- Abgabe der Calmodulin Konstrukte mit 2, 4 und 6 Glycinen Linker zum Sequenzieren; Es wurde hierfür immer Klon Nr 1 abgegeben; Auswertung sollte nach Möglichkeit bitte jemand anders machen, da ich ab morgen auf Urlaub bin;)<br>
+- deliverated the constructs with 2, 4 and 6 glycines linker to GATC for sequencing; i used clone Nr 1 of each<br> construct; Natalia  analyzed the sequences for me because i was on holiday ;)
-<p>Natalia, Dario (13 - 16)<br>
+<p>Natalia, Dario (13 - 16 o´clock)<br>
 - Amplification of the dhfr1_wz_dhfr2 clone<br>
 - There were 6 large and 6 small clones colonies selected from the plate with
@@ Line 376: / Line 379: @@
 == Tu, 21th August 2007 ==
-Moritz (12-20.30):<br>
+Moritz (12-20.30 o´clock):<br>
 - PCR for Phytochrome and FHY1 Inserts
-Dario (12-20:30):<br>
+Dario (12-20:30 o´clock):<br>
 - Expressions culture of b1_calmo_b2 Gly 4 (XL-1)<br>
-Natalia (9:30 - 21:00)<br>
+Natalia (9:30 - 21:00 o´clock)<br>
-- Preppen der gepickten Klone vom 20/08/07<br>
+- spin prep from picked clones (08/20/07)<br>
-- von allen (12) Klonen wurde ein Glycerinstock angeleget<br>
+- of all clones(12)glycerin stocks were created <br>
-- Klone 3, 5, 6, 7, 9, 10 wurden runterzentrifugiert und eingefroren<br>
+- clones 3, 5, 6, 7, 9, 10 were centrifuged and frozen <br>
-- Klone 1, 2, 4, 8, 11, 12 wurden geprept und testverdaut mit EarI<br>
+- spin prep and analytic digestion with EarI of clones 1, 2, 4, 8, 11, 12 <br>
-- Gel laufen lassen mit dem Testveradu<br>
+- Gel electrophoresis  of analytic digestion<br>
-- Präparativen Verdau mit Klon2 angesetzt<br>
+- preparative digestion of clone 2 <br>
-- Platten für den Wachstumstest geggossen<br>
+- Prepared plates for the growth test<br>
-- Plasmidkarte für das Plasmid: dFR320d_dhfr1_wz_dhfr2A erstellt<br>
+- Made a plasmif map for the plasmid: dFR320d_dhfr1_wz_dhfr2A <br>
 == We, 22th August 2007 ==
@@ Line 399: / Line 402: @@
 -Overnight culture of DHFR plasmids from laboratory glycerin stocks<br>
-Dario (9.30-21.00 Uhr):<br>
+Dario (9.30-21.00 o´clock):<br>
 - Transformation of b1_calmo4,4_b2 clone 1 in RV308<br>
 - Purification of the expression culture of b1_calmo4,4_b2 Klon 1 in XL-1<br>
 - Preparation of the buffers for the periplasmatic extraction of the proteins of b1-cal4,4_b2 K1<br>
-Natalia (9:00 - 15:00)<br>
+Natalia (9:00 - 15:00 o´clock)<br>
-- Sequenzierung von dhfr1_wzdhfr2 Klon 1/4<br>
+- Sequencing of dhfr1_wz_dhfr2 clon 1/4<br>
-- Präparativer Verdau v.:21/08/07 Bande ausgeschnitten<br>
+- gel electrophoresis of preparative digestion (08/21/07) <br>
-- Präparativen Verdau mit Klon1/4 angesetzt<br>
+- preparation of prep. digestion of clone 1/4 <br>
-- Klon1/2/4 von dhfr1_wz_dhfr2 ÜNK angesetzt<br>
+- overnight culture of clones 1/2/4 (dhfr1_wz_dhfr2) <br>
-- ÜNK von b1_2,4,6calmo246_b2 angesetzt<br>
+- overnight culture of b1_2,4,6calmo246_b2 <br>
-- Auswertung der Sequenzierung vom 10/08/07 v. dhfr2<br>
+- Analysis of the sequencing (08/10/07) dhfr2<br>
 == Thu, 23th August 2007 ==
-<p><b>Group meeting </b>(14:30 bis 16:00 Uhr)<br>
+<p><b>Group meeting </b>(14:30 bis 16:00 o´clock)<br>
 </p>
 <p><b>1. With pFR320p_b1_2,4,6_b2</b><br>
@@ Line 423: / Line 426: @@
 &nbsp;&nbsp;&nbsp; - Transformation in BL21 with pREP4<br>
 </p>
-<p><b>2. Bei DHFR1_wz_DHFR2</b><br>
+<p><b>2. With DHFR1_wz_DHFR2</b><br>
 &nbsp;&nbsp;&nbsp; - Sequencing: Exchanges in a few bases, but not in&nbsp; DHFR.
 Possible mistake: Plasmid is good, the problem is the plasmid map<br>
@@ Line 435: / Line 438: @@
 </p>
 <p><b>4. PCR</b><br>
-&nbsp;&nbsp;&nbsp; - With Fhy1, Phy A and Fhy is everything working (Digestions
+&nbsp;&nbsp;&nbsp; -  everything is working out with Fhy1, Phy A and Fhy  (Digestions
 and PCR)<br>
-&nbsp;&nbsp;&nbsp; - Now must Fhy1 with YFP/YFP with FHY/ PHy A with CFP merged<br>
+&nbsp;&nbsp;&nbsp; - Now  Fhy1 with YFP/YFP with FHY/ PHy A with CFP must be merged<br>
 &nbsp;&nbsp;&nbsp; - Next experiments. FRET ( Activity test)<br>
 </p>
 <p><br>
-Dario (10.30-21.30 Uhr):<br>
+Dario (10.30-21.30 o´clock):<br>
 - Transformation b1_calmo4,4_b2 clone 1 in RV308 was repeated.<br>
 - Purification of the expression culture of b1_calmo4,4_b2 clone 1 in XL-1 still
 in process.<br>
-- Assinting Natalia with the Calmo-constructs with different concentration
+- Assisting Natalia with the Calmo-constructs with different concentration
 of&nbsp; Ca2+, IPTG und Amp</p>
-Natalia (9.30-21.30)<br>
+Natalia (9.30-21.30 o´clock)<br>
-- Auswerten der Plasmidsequenzierungen von dhfr1_wz_dhfr2 Klon4 und Klon1<br>
+- Analysis of the sequencing of dhfr1_wz_dhfr2 clone 4 and clone 1<br>
-- Gel laufen lassen mit d. Präparativen Verdau von dhfr1_wz_dhfr2<br>
+- gel running with the preparative digestion (dhfr1_wz_dhfr2)<br>
-- Vektorbande v. Klon4 ausgeschnitten, dephosphoryliert und eingefroren<br>
+- vector of clone 4 was dephosphorylated and frozen<br>
-- Messen der OD der UNK von b1-2,4,6calmo2,4,6-b2 immer wieder verdünnt und anschließend auf Platten mit unterschiedlichen Konzentrationen (Calcium und Amp) ausgestrichen<br>
+- OD of the overnight culture ofb1-2,4,6calmo2,4,6-b2 was always diluted and crossed out on plates with different     concentrations <br>
 Moritz (9.30 - 16.30 o´clock):<br>
@@ Line 469: / Line 472: @@
 - new ligation of the Fhy1-YFP constructs, as well as PhyA-CFP<br>
-Dario (12-16 Uhr):<br>
+Dario (12-16 o´clock):<br>
 - Counting of colonies on plates of bl_calmo2,4,6_b2 with different concentrations of Ca2+,<br>  Amp und IPTG.
-- PheBo loding buffer for the protein purification of b1_calmo4_b2 in XL-1 was changed.<br>
+- PheBo loading buffer for the protein purification of b1_calmo4_b2 in XL-1 was changed.<br>
-Natalia (9.00-11.00)<br>
+Natalia (9.00-11.00 o´clock)<br>
-- erneut: Präparatiververdau von dhfr1-wz-dhfr2 Klon4<br>
+- again: preparative digestion of dhfr1-wz-dhfr2 clone 4<br>
-- dhfr1-wz-dhfr2 Klon1 und 2 wurden geprept und eingefroren<br>
+- spin prep of dhfr1-wz-dhfr2 clone 1 and 2 <br>
-- Auswertung der ausplattierten Klone b1-2,4,6calmo2,4,6-b2<br>
+- evaluation of the clones b1-2,4,6calmo2,4,6-b2<br>
 == Sa, 25th August 2007 ==
@@ Line 493: / Line 496: @@
 - Ligation: Fhy1(big and small)-YFP; YFP-Fhy1(big and small); PhyA-CFP<br>
-Natalia, Corinna(9.00 - 16.00)<br>
+Natalia, Corinna(9.00 - 16.00 o´clock)<br>
-- gel: präparativer Verdau vom 24/08/07 ausgeschnitten und dephosphoryliert<br>
+- Gel: preparativ digestion from 24/08/07 was cut and dephosphorylated<br>
-- Sequenzierungen ausgewertetvon b1-2,4,6calmo2,4,6-b2<br>
+- Analysis of the sequence of b1-2,4,6calmo2,4,6-b2<br>
 == Tu, 28th August 2007 ==
 Corinna, Natalia (9.30-22.30 o´clock):<br>
-- Auswertung der Sequenzierungen für die Lactamase-Calmodulin-Konstrukte (Linkerlänge 2,4,6 AS): viele Fehler im Calmodulin, Veilleicht aber auch nur Fehler in der Sequenzierung<br>
+- Analysis of the sequences for the lactamase-Calmodulin-constructs (Linker-length 2,4,6 AS): too many problems with Calmodulin, probably just mistekes On the sequencing process<br>
-- neue Proben zur Sequenzierung mit neuem Primer ca. 80 bp vorm Calmodulin abgegeben<br>
+- New assays from Calmodulin to be sequenced were given<br>
-- neue PCR für Calmodulin mit verschiedenen Linkerlängen; Reinigung der PCR-Produkte<br>
+- new PCR for Calmodulin with different linker-lengths; purification of the PCR products<br>
-- Verdau der PCR-Produkte mit KpnI und SpeI<br>
+- Digestion of the PCR-products with KpnI and SpeI<br>
-- prep. Gel : Template-Banden waren zu erkennen, aber leider keine Produkt<br>
+- prep. Gel : Template-Bands were to see, but sadly no product<br>
-- erneuter Ansatz der PCR; diesmal wurden die Vorverdünnungen von Primern und Template frisch hergestellt<br>
+- New preparations of PCR; but these time were the attenuate examples of Primer and Template fresh<br>
 Moritz (11-22.30 o´clock):<br>
@@ Line 520: / Line 523: @@
 - Spin Prep of the overnight culture (Fhy1(big and. small)-YFP; YFP-Fhy1(big and small); PhyA-CFP)<br>
-Natalia, Corinna (9.30-16.00)<br>
+Natalia, Corinna (9.30-16.00 o´clock)<br>
-- Reinigung der PCR-Produkte<br>
+- Purification of the PCR-products<br>
-- analytisches Gel laufen lassen<br>
+- analitic gel was done<br>
-- Verdau der PCR-Produkte und Präparatives Gel<br>
+- Digestion of the PCR-products and preparativ gel<br>
-- Ligationsansatz<br>
+- Ligation assay<br>
 == Do, 30th August 2007 ==
@@ Line 534: / Line 537: @@
 - Design of plasmid maps (Fhy1(big and small)-YFP; YFP-Fhy1(big and small); PhyA-CFP)<br>
-Corinna, Natalia (9.30-11.30)<br>
+Corinna, Natalia (9.30-11.30 o´clock)<br>
-- Transformation der Ligation dhfr1-2,4,6calmo2,4,6-dhfr2 ; b1-2,4,6calmo2,4,6-b2<br>
+- Transformation of the Ligation dhfr1-2,4,6calmo2,4,6-dhfr2 ; b1-2,4,6calmo2,4,6-b2<br>
 == Fr, 31th August 2007 ==
@@ Line 543: / Line 546: @@
 - Analysis of the Sequencing (Fhy1(big bzw. small)-YFP; YFP-Fhy1(big and small); PhyA-CFP):<br>
-Corinna, Natalia (10-12)<br>
+Corinna, Natalia (10-12 o´clock)<br>
-- Amplifikation gepickter Klone<br>
+- Amplification of the clones<br>
 == Sa, 1st Sept.2007 ==
-Natalia (14.30-17.30)<br>
+Natalia (14.30-17.30 o´clock)<br>
-- Glycerinstock d. amplifiz. Klone<br>
+- Glycerine stock of the amplified clones<br>
-- Klone 1,2 von dhfr1-2,4,6calmo2,4,6-dhfr2 b1-2,4,6calmo2,4,6-b2 geprept<br>
+- spin prep of clone 1,2 of dhfr1-2,4,6calmo2,4,6-dhfr2 b1-2,4,6calmo2,4,6-b2 <br>
-- klon 3 von dhfr1-2,4,6calmo2,4,6-dhfr2 b1-2,4,6calmo2,4,6-b2 runterzentrifugiert und eingefroren<br>
+- clone 3 of dhfr1-2,4,6calmo2,4,6-dhfr2 b1-2,4,6calmo2,4,6-b2 were centrifuged and frozen<br>
 == Mo, 3rd Sept. 2007 ==
-Natalia (10.30-19.00)<br>
+Natalia (10.30-19.00 o´clock)<br>
-- Plasmidkarten für dhfr1-2,4,6calmo2,4,6-dhfr2 erstellet<br>
+- Creating plasmid maps of dhfr1-2,4,6calmo2,4,6-dhfr2 <br>
-- Testverdau: dhfr1-2,4,6calmo2,4,6-dhfr2 <br>
+- analytic digestion: dhfr1-2,4,6calmo2,4,6-dhfr2 <br>
-- Miniprep: dhfr1-6calmo6-dhfr2 Klon3, b1-2,4,6calmo2,4,6-b2 Klon3<br>
+- spin prep: dhfr1-6calmo6-dhfr2 clone 3, b1-2,4,6calmo2,4,6-b2 clone 3<br>
-- Sequenzierungen vom 01.09.07 ausgewertet<br>
+- Analysis of the Sequencing from 09.01.07 <br>
-- zum Sequenzieren abgegeben: dhfr1-2,4calmo2,4-dhfr2 Klon1,2<br>
+- sequencing: dhfr1-2,4calmo2,4-dhfr2 clone 1,2<br>
 == Dienstag, 4th Sept. 2007 ==
-Natalia (10.30-19.30)<br>
+Natalia (10.30-19.30 o´clock)<br>
-- Testverdau für b1-2,4,6calmo2,4,6-b2 dhfr1-6calmo6-dhfr2 Klon3 angesetzt, laufen lassen und ausgewertet<br>
+- analytic digestion of b1-2,4,6calmo2,4,6-b2 dhfr1-6calmo6-dhfr2 clone 3, gel electrophoresis and analysing<br>
-- erneute Ligation mit b1-2,4,6calmo2,4,6-b2 angesetzt<br>
+- again: Ligation of b1-2,4,6calmo2,4,6-b2 <br>
-- Präparativer Verdau: b1-wz-b2 angesetzt, da Vektor fast leer ist<br>
+- preparative digestion: b1-wz-b2, because vector is almost empty <br>
-- mein Hirn mit Sequenzierungen gequält (Auswertung von dhfr1-2,4calmo2,4-dhfr2 Klon2,1)<br>
+- torturing my brain with sequencing (Sequence analysis dhfr1-2,4calmo2,4-dhfr2 clones 2,1)<br>
-- zum Sequenzieren abgegeben: dhfr1-6calmo6-dhfr2 klon3, dhfr1-4calmo4-dhfr2 Klon1<br>
+- delivering sequencing: dhfr1-6calmo6-dhfr2 clone 3, dhfr1-4calmo4-dhfr2 clone 1<br>
-- für Phillipp: yfp big und small amlifiziert: je 5 Klone<br>
+- for Philipp: amplification of yfp big and small (5 clones each) <br>
 == Mittwoch, 5th Sept. 2007 ==
-Natalia (10.00-15.00)<br>
+Natalia (10.00-15.00 o´clock)<br>
-- Transformation der Ligation von b1-2,4,6calmo2,4,6-b2 in XL-1<br>
+- transformation of  b1-2,4,6calmo2,4,6-b2- ligation into XL-1<br>
-- neue Platten mit Cmp gegossen<br>
+- made fresh  Cmp-plates <br>
-- parallel zur Ligation wurden 8 weitere Klone (Trafo vom 31.08.07) von b1-2,4,6calmo2,4,6-b2 gepickt<br>
+-picked further 8 clones with b1-2,4,6calmo2,4,6-b2 (transformated: 31 Aug.07)parallel to new ligation<br>
-- Ptäparatives Gel (b1-wz-b2 - Vektorverdau) auf dem Gel aufgetragen, die Banden ausgeschnitten und gereinigt<br>
+-running of preparative gel with b1-wz-b2 - vector digest, isolation and purification of bands<br>
 Philipp (17.30-21.00) <br>
 - glycerol-stocks with YFP-Fhy1 (5x "big", 5x "small")<br>
@@ Line 584: / Line 587: @@
 == Do, 6th Sept. 2007 ==
-Natalia (10.00-18.30<br>
+Natalia (10.00-18.30 o´clock)<br>
-- Glycerinstock von den amplifizierten Klonen (5.9.7) angelegt, die ersten vier Klone wurden geprept, die anderen vier nur als Pellett eingefroren<br>
+- creating glycerinstocks of amplified clones (5.9.7) , spin prep of the first four clones,the other four were frozen as a pellett<br>
-- geprepten Klone (1-4) b1-2,4,6calmo2,4,6-b2 wurden mit Kpn1 und Spe1 verdaut und aufs Gel aufgetragen<br>
+- analytic digestion with KpnI and SpeI of (clones 1-4) b1-2,4,6calmo2,4,6-b2 <br>
-- b1-2calmo2-b2 Klon 2 und b1-6calmo6-b2 Klon 4 hatten nach der Größe zu urteilen das richtige Insert und wurden zum Sequenzieren abgegeben<br>
+- gel electrophoresis of analytic digestion<br>
-- Trafo vom 05/09/07: von den ligierten Plasmiden wurden je 10 Klone gepickt und amplifiziert (b1-2,4,6calmo2,4,6-b2)<br>
+- b1-2calmo2-b2  clone 2 and b1-6calmo6-b2 clone 4 had the right insert. Sequencing were made.<br>
+- Transformation of 09/05/07: of the ligated plasmids were 10 clones picked and amplified (b1-2,4,6calmo2,4,6-b2)<br>
 == Fr, 7th Sept. 2007 ==
-Natalia (10.00-18.00)<br>
+Natalia (10.00-18.00 o´clock)<br>
-- Vektor b1-wz-b2 vom 05/09/07 dephosphoryliert<br>
+- dephosphorylation of  b1-wz-b2 from 09/05/07 <br>
-- Gepickte Klone vom 06/09/07: von allen 30 (b1-2,4,6calmo2,4,6-b2)Klonen wurde ein Glycerinstock angelegt, Klon 1-5 b1-2,4,6calmo2,4,6-b2 wurde geprept und testverdaut, Klon 6-10 als Pellett eingefroren<br>
+- picked clones from 09/06/07: glycerol stock of all 30 clones (b1-2,4,6calmo2,4,6-b2)<br>
-- nach dem Testverdau: 3 von fünf Klonen (b1-2,4,6calmo2,4,6-b2) sind positiv<br>
+- spin prep and analytic digestion of clones 1-5 of b1-2,4,6calmo2,4,6-b2<br>
+- clones 6-10 of b1-2,4,6calmo2,4,6-b2 were frozen as a pallett<br>
+- after analytic digestion: 3 of 5 clones were positive (b1-2,4,6calmo2,4,6-b2) <br>
 == Mo, 10th Sept. 2007 ==
-Natalia (10.00-17.00)<br>
+Natalia (10.00-17.00 o´clock)<br>
-- angefallene Sequenzierungen ausgewertet<br>
+- analyzing sequencing<br>
-- Klon 1 von b1-2,4,6calmo2,4,6-b2 vom 07/09/07 zum Sequenzieren abgegeben<br>
+- clone 1 of b1-2,4,6calmo2,4,6-b2 from 09/07/07 were delivered for sequencing<br>
-Philipp (13.30-16.30)<br>
+Philipp (13.30-16.30 o´clock)<br>
-- Abgabe der am 05.09.geprepten Plasmide zur Sequenzierung<br>
+- plasmids from 09.05.07 were delivered for sequencing <br>
-- Einlesen, Erkundigen...<br>
+- read in, getting information...<br>
 == Di, 11th Sept. 2007 ==
-Philipp (11.30-16.00)<br>
+Philipp (11.30-16.00 o´clock)<br>
 - evaluated sept.10th´s sequencing results<br>
 == Mi, 12th Sept. 2007 ==
-Philipp (12.00-15.30)<br>
+Philipp (12.00-15.30 o´clock)<br>
 -  evaluated sequencing (30./31. Aug) results .  <br>
 - generated plasmid/fragment-maps <br>
@@ Line 622: / Line 626: @@
 == Do, 13th Sept. 2007 ==
-Philipp (13.00-16.30)<br>
+Philipp (13.00-16.30 o´clock)<br>
--mehr maps erstellt, Sequenzierungen nochmal genauer betrachtet<br>
+-creating more plasmid maps, analyzing sequences much more exactly <br>
--10 neue Klone mit (hoffentlich) PhyA-CFP-Plasmid gepickt und als ÜN-Kulturen (1-10) angesetzt<br>
+-10 new clones with PhyA-CFP-Plasmid were picked
--ÜNK von Glycerinstock (von Urs) mit CFP-Ausgangsplasmid angesetzt<br>
+-overnight culture (clones 1-10) were  started <br>
+-overnight cultures of glycerol stock (Urs) with CFP-plasmid were started<br>
 == Fr, 14th Sept. 2007 ==
-Philipp (10.30-18.00)<br>
+Philipp (10.30-18.00 o´clock)<br>
--ÜNK´s 1,3,5,7,9 sowie CFP-Ausgangsplasmid geprept<br>
--eigenen Glycerinstock mit CFP-Plasmid angelegt<br>
+-spin prep of the over night culturs 1,3,5,7,9 and CFP-starting plasmid <br>
--ÜNK´s 2,4,6,8,10 abzentrifugiert und eingefroren <br>
+-glycerol stocks with CFP-Plasmid<br>
--Verdau (HindIII) und Analysegel für o.g. Preps um Einbau von PhyA zu prüfen:<br>
+-over night cultures 2,4,6,8,10 centrifuged and frozen <br>
-positiver Befund für Klone 3,7,9<br>
+-digestion (HindIII) and analytic gel for the spin preps to proof the integration from PhyA:<br>
--diese zum Sequenzieren abgegeben<br>
+positive for clone 3,7,9<br>
+-probes delivered for sequencing<br>
 == Sa, 15th Sept. 2007 ==
-Philipp(14.00-18.30)<br>
+Philipp(14.00-18.30 o´clock)<br>
--Präparativer Verdau des CFP-Ausgangsplasmids (Vektors) mit EcoRI & SpeI
+- preparative digestion of the CFP-plasmid (vector) with EcoRI and SpeI
 == So, 16th Sept. 2007 ==
-Philipp (16.00-18.30)<br>
+Philipp (16.00-18.30 o´clock)<br>
--Prep-Gel gegossen und mit Verdau vom 15.09. (CFP-Vektor) laufen lassen; leider keine <br>
-Bande zu erkennen (nur "Schmier",Konzentration wohl zu gering/"Star-Aktivität"?...)<br>
+-running of a preparative gel with the CFP-Vektor; unfortunately no prominent bands; maybe due to "star-activity" or contamination?<br>
--Sequenzierungen vom 14.09. (PhyA-CFP...) ausgewertet; erster Eindruck nicht schlecht...<br>
+-received sequences of sept. 14th (PhyA-CFP...); first impression not too bad...<br>
 == Mo, 17th Sept. 2007 ==
-Philipp(12.00-17.00)<br>
+Philipp(12.00-17.00 o´clock)<br>
--Testverdau/Analysegel vom 14.09. (PhyA-CFP mit HindIII) wiederholt, positiv<br>
--betroffene Proben (3,7,9) nochmal mit pQE-RP sequenzieren lassen<br>
+-preparative digestion /analytic gel (09.14.2007) -PhyA-CFP mit HindIII- repeated, positiv<br>
--Planung, Ansatz der Vorkultur für eine Expressionskultur mit dhfr1-4/6calmo4/6-dhfr2<br>
+-sequencing of the concerned probes (3,7,9) again with pQE-RP<br>
+-planning; approach of a cultur for the expression cultur with dhfr1-4/6calmo4/6-dhfr2<br>
 == Di, 18th Sept. 2007 ==
-Philipp(10.00-21.00)<br>
+Philipp(10.00-21.00 o´clock)<br>
--Expressionskultur (RV308 mit dhfr1-4/6-calmo-4/6-dhfr2) hochgezogen, abzentrifugiert, eingefroren<br>
--Wachstums-Vortest (s.o.) in M9-Minimalmedium angesetzt (M9; M9 mit TMP; M9 mit TMP+CaCl)<br>
+-expression cultur (RV308 with dhfr1-4/6-calmo-4/6-dhfr2), centrifuged and frozen<br>
+-growth test (see above) in M9 medium (M9; M9 with TMP; M9 with TMP+CaCl)<br>
 == Mi, 19th Sept. 2007 ==
-Philipp(11.00-16.00)<br>
+Philipp(11.00-16.00 o´clock)<br>
--Auswertung der RP-Sequenzierung von PhyA-CFP vom 17.09.; offenbar scheint PhyA am<br>
-N-Terminus mit CFP ligiert zu sein... ->Neuansatz einer 10ml ÜNK mit CFP-Glycerinstock von Urs<br>
+-analysis of the RP-sequencing from PhyA-CFP (09.17.2007); obviously PhyA is ligated at the N-terminus with CFP<br>
--Protokoll/Graph/Journaleintrag zur Expressionskultur vom 28.09.<br>
+-> new approach of a 10ml over night culture with CFP glycerol stock from Urs<br>
+-protocol/graph/Journal entry: expressions cultur (09.28.2007)<br>
 == Do, 20th Sept. 2007 ==
-Philipp(10.30-20.00)<br>
+Philipp(10.30-20.00 o´clock)<br>
--Spinprep von pAR200-cJun-CFP<br>
--iGEM-Vortrag gehört<br>
+-Spinprep from pAR200-cJun-CFP<br>
--SDS-Gel mit dhfr-4/6calmo laufen lassen, ge- und entfärbt, gescant<br>
+-iGEM lecture<br>
--OD der M9-Kulturen gemessen -> Vortest gibt Anlass zur Hoffnung auf Funktion...<br>
+-running of the SDS gel with dhfr-4/6calmo, staining, scan<br>
--Besprechung (Jamboree, Arbeitszeiten)<br>
+-measurement of the OD from the M9-cultures -> preparative test makes hope...<br>
+-meeting(themes: Jamboree, working times)<br>
 == Fr, 21th Sept. 2007 ==
-Philipp(11.00-12.30)<br>
+Philipp(11.00-12.30 o´clock)<br>
--SDS-Gel (jetzt fertig entfärbt) gescant<br>
--nach langer Suche alle dhfr-Daten im blac-Ordner gefunden!?<br>
+-SDS gel (now ready discolored) scaned<br>
--dann Peptidinfo-Sheet generiert<br>
+-peptide-information sheet generated<br>
 == So, 23th Sept. 2007 ==
-Philipp(14.30-20.00)<br>
+Philipp(14.30-20.00 o´clock)<br>
--Verdau(EcoRI,SpeI)des CFP-Vektors wiederholt, Gel (5 Banden statt 2!?), Extraktion<br>
--Ansatz einer neuen ÜNK für dieses Plasmid (Glycerinstock von Urs, diesmal ein anderer...)<br>
+-digestion(EcoRI,SpeI) of the CFP vectors repeated, gel extraktion (only 5 bands instead of 2!?)<br>
--Ansatz eines weiteren Wachstums-Vortests (bzw. der Gegenprobe mit dhfr-winzip) in M9 für dhfr1-4calmo4-dhfr2<br>
+-approach of an new over night culture for this plasmid (glycerol stock from Urs)<br>
+-approach of another growth test (cross-check with the dhfr-winzip construct for "background"-measurement) in M9 for dhfr1-4calmo4-dhfr2 construct<br>
 == Mo, 24th Sept. 2007 ==
-Philipp(11.00-20.00)<br>
+Philipp(11.00-20.00 o´clock)<br>
--Puffer für Proteinreinigung angesetzt<br>
+-preparation buffer for the protein purification<br>
 -dhfr1-6calmo6-dhfr2 fraktioniert über Ni-NTA-Säule gereinigt<br>
--ÜNK mit pAR200-JunW-CFP abzentrifugiert...<br>
+-over night culture with pAR200-JunW-CFP centrifuged...<br>
--OD der gestern angesetzten M9-Kulturen gemessen->Hoffnung wächst...<br>
+-measurement of the OD of the M9-cultures ->The hope is growing...<br>
 == Di, 25th Sept. 2007 ==
-Philipp(09.00-14.30)<br>
+Philipp(09.00-14.30 o´clock)<br>
--SDS-Gel mit gereinigtem Protein (dhfr-4calmo..; vom 24.09.) laufen lassen<br>
--Wachstums-Vortest in M9 bei verschiedenen Ca2+-Konzentrationen angesetzt<br>
+-running of a SDS gel with the purificated protein (dhfr-4calmo..; 24.09.)<br>
--ÜNK von RV308 mit dhfr1-4calmo4-dhfr2 angesetzt<br>
+-first growth tests in M9 at different Ca2+-concentrationes<br>
+-preparation of the over night culture from RV308 with dhfr1-4calmo4-dhfr2<br>
 == Mi, 26th Sept. 2007 ==
-Philipp(12.30-17.00)<br>
+Philipp(12.30-17.00 o´clock)<br>
--OD´s der M9-Hintergrund-Testkulturen vom 23.09. gemessen->wie´s aussieht kein Hintergrund!!<br>
--dhfr-4calmo-ÜNK geprept<br>
+-measurement of the ODs from the M9-background cultures (23.09.)->it looks like there's no background!!<br>
--SDS-Gel ausgewertet, gescant<br>
+-spin prep of the dhfr-4calmo over night culture<br>
--Peptidinfo´s berichtigt...<br>
+-SDS gel evaluated and scaned<br>
+-corrected peptide-informations...<br>
 == Do, 27th Sept. 2007 ==
-Philipp(14 Uhr)<br>
+Philipp(14 o´clock)<br>
--OD´s der M9-Kulturen vom 23. sowie 25.09.gemessen(bestes Wachstum bisher bei 1,6mM Ca2+)<br>
+-measurement of the ODs der M9-Kulturen from 23.9 and 25.09.07;<br>
+best growth at 1,6 mM Ca2+ concentration<br>
 == Mo, 01st Okt. 2007 ==
-Philipp(11.00-17.30)<br>
+Philipp(11.00-17.30 o´clock)<br>
--Verdau (KpnI/SpeI),Gel,Extraktion,Konz.best. von Calmodulin (Insert) und blac1-winzip-blac2(Vektor)<br>
--Neuansatz der M9-Ca2+-Verdünnungsreihe<br>
+-Digestion with KpnI/SpeI, gel running, gel extraktion, determination of the concentration from Calmodulin (Insert) and blac1-winzip-blac2(Vektor)<br>
+-new approach of the M9-Ca2+ dilution series<br>
 == Di, 02nd Okt. 2007 ==
-Philipp(...)<br>
+Philipp<br>
--OD´s der M9-Kulturen vom 01.10. gemessen<br>
+-measurement of the ODs of the M9-cultures (01.10.)<br>
 == Mi, 03rd Okt. 2007 ==
-Philipp(16.00-19.00)<br>
+Philipp(16.00-19.00 o´clock)<br>
--weiter OD´s der M9-Kulturen vom 01.10. gemessen, klarer Zusammenhang zwischen<br> Ca2+-Konzentration und Wachstum zu erkennen->"dhfr-4calmo" scheint zu funktionieren!<br>
--Maps für die Synthese der dhfr1-4calmo4-dhfr2 iGEM-parts erstellt<br>
+-more measurements of the ODs from the M9-cultures (01.10.07); clear link between Ca2+-concentration and growth recognized<br>
+'''->"dhfr-4calmo-construct" seems to work!'''<br>
+-Maps for the synthesis for the dhfr1-4calmo4-dhfr2 iGEM-parts created<br>
 == Do, 04th Okt. 2007 ==
-Philipp(12.30-21.30)<br>
+Philipp (12.30-21.30 o´clock)<br>
--immer noch OD´s gemessen<br>
+-Measurements of the OD<br>
--Sequenzen für die Synthese fertig gemacht.<br>
+-Sequences for the synthesis prepared<br>
+'''''...At that point of time we had to leave our -not really finished- lab-work and focus on the theoretical planing and uoploading of our parts and their synthesis by GeneArt...'''''<br>
 [https://2007.igem.org/Freiburg BACK]

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