Revision as of 18:21, 26 October 2007 by Maximilian (Talk | contribs)

There are days left until the Jamboree!


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.
    - 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
    - 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: ???

Planning and doing the digetion of dFR320dp-blac1-winzip-blac2, these time with BssSI
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
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
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 concentrations( 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
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
- 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 und 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

    - 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 over night 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 Uhr):
- 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)
- 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 Uhr)

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

Dario (10.30-21.30 Uhr):
- 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.
- Assinting Natalia with the Calmo-constructs with different concentration of  Ca2+, IPTG und Amp

Natalia (9.30-21.30)
- 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 Uhr):
- Counting of colonies on plates of bl_calmo2,4,6_b2 with different concentrations of Ca2+,
Amp und IPTG. - PheBo loding buffer for the protein purification of b1_calmo4_b2 in XL-1 was changed.

Natalia (9.00-11.00)
- 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)
- 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)
- 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)
- 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)
- Amplifikation of the clones

Sa, 1st Sept.2007

Natalia (14.30-17.30)
- Glycerinstock 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)
- 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)
- 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 : each 5 clones

Mittwoch, 5th Sept. 2007

Natalia (10.00-15.00)
- Transformation der Ligation von b1-2,4,6calmo2,4,6-b2 in XL-1
- neue Platten mit Cmp gegossen
- parallel zur Ligation wurden 8 weitere Klone (Trafo vom 31.08.07) von b1-2,4,6calmo2,4,6-b2 gepickt
- Ptäparatives Gel (b1-wz-b2 - Vektorverdau) auf dem Gel aufgetragen, die Banden ausgeschnitten und gereinigt
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
- 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)
- 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)
- 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)
- plasmids from 09.05.07 were delivered for sequencing
- read in, getting information...

Di, 11th Sept. 2007

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

Mi, 12th Sept. 2007

Philipp (12.00-15.30)
- 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)
-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)

-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


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

So, 16th Sept. 2007

Philipp (16.00-18.30)

-running of a preparative gel gegossen und mit Verdau vom 15.09. (CFP-Vektor) laufen lassen; leider keine
Bande zu erkennen (nur "Schmier",Konzentration wohl zu gering/"Star-Aktivität"?...)
-Sequenzierungen vom 14.09. (PhyA-CFP...) ausgewertet; erster Eindruck nicht schlecht...

Mo, 17th Sept. 2007


-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


-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


-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


-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


-SDS gel (now ready discolored) scaned
-peptidinformation sheet generated

So, 23th Sept. 2007


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

Mo, 24th Sept. 2007


-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


-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


-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
-peptidinformations corrected...

Do, 27th Sept. 2007

Philipp(14 Uhr)

-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


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

Di, 02nd Okt. 2007


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

Mi, 03rd Okt. 2007


-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)

-Measurements of the OD
-Sequences for the synthesis prepared