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Labjournal Cellfree August

Thu, 27.08.2015

PCR to amplify HA_GFP_HisHis for surface immobilization (Ramona)

GFP was amplified from PCR product aqSK (with the appropriate binding sites; from AG Roth) with an amino-labeled reverse primer (qSK11) and a Cy3-labeled forward primer (qSK13)

Component	Volume [µl]
aqSK	0.5
qSK11	2
qSK13	2.5
Phusion High GC Buffer	10
Phusion polymerase	0.5
dNTPs	2.5
ddH2O	32

3fold master mix was prepared and divided into 3 PCR tubes
cycling conditions were chosen according to the lab journal entry from 16.8.15 (Luisa)
PCR product was cleaned-up with the PCR purification kit from Qiagen
result: ~60 µl DNA with a concentration of 66.1 ng/µl
10 µl of the PCR product were analyzed on a 1 % agarose gel: amplification of GFP was successful (image could not be saved due to the scanner)

Wed, 26.08.2015

Expression of HA-GFP-His6-His6 on NiNTA slide

(Luisa)

HA-GFP-His6-His6 was expressed in 15 µl reactions
2 reactions were performed as usual in a 1.5 ml tube
6 reactions were performed by using a mastermix on a NiNTA slide in spots (2 slides with 3 reactions each)
4 reactions were performed without DNA on a NiNTA slide in spots (2 slides with 2 reactions each)
all samples were incubated for 3 hours at 37°C with the slide being covered by parafilm instead of a microsope slide
after expression the slides will be incubated at 4°C over night and then measured in the iRIf
for more results please refer to SurChem lab journal

Tue, 25.08.2015

Expression of HA-GFP-His6-His6 on NiNTA slide

(Luisa)

HA-GFP-His6-His6 was expressed in 15 µl reactions
2 reactions were performed as usual in a 1.5 ml tube
6 reactions were performed by using a mastermix on a NiNTA slide in spots (2 slides with 3 reactions each)
4 reactions were performed without DNA on a NiNTA slide in spots (2 slides with 2 reactions each)
all samples were incubated for 3 hours at 37°C with the slide being covered by a regular microscope slide
after expression the slides will be incubated at 4°C over night and then measured in the iRIf
for more results please refer to SurChem lab journal

when removing the microsope slide, the spots converged. The iRIf measurement will be performed anyway, but the entire experiment is repeated for distinct spots.

Mon, 24.08.2015

Maxi of LUC Plasmid from Promega and of pIG_1104 with Promega Kit

(Sabi)

Elution in 750 µl of nuclease free water
Luc: 560 ng/µl
Tetanus:

Expression of pIG_1104 (tetanus)

(Koko)

50µl rx's, 37°C, 2h, 0rpm
- 3x KK + 4,75µl pIG_1104 DNA (=2µg)
- 3x BK + 4,75µl pIG_1104 DNA (=2µg)
- 2x KK + 4,75µl water (neg)
- 2x BK + 4,75µl water (neg)

Sun, 23.08.2015

Concentration of maxi prep of HA-GFP-His6-His6 and pBEStluc

(Luisa)

5 columns per sample, elution in 12 µl each, pooled for measurement

Ponceau staining of expression of various GFPs

(Luisa)

Sat, 22.08.2015

Western blot of expression of various GFPs

(Luisa)

blots are so bad they have to be repeated anyways. therefore i did not waste any time making the pictures pretty..

Fri, 21.08.2015

Repetition of variation of MgOAc start concentration in triplicates

(Koko)

finally came round to repeat critizised experiment
- 20 µl rx's
- 800 ng pBESTluc DNA (=2000 ng/50µl)
- variation from 12 mM to 16 mM of MgOAc, as first experiment suggested 14 mM to be the optimum
- 2 h, 37°C, 0 rpm
- adding 20 µl Dilution agent + 50 µl Luciferase Assay reagent (immediately before platereading)

plate layout

	1	2	3	4	5	6	7
D	12mM	13mM	14mM	15mM	16mM	neg.(+Rmix)	neg.(+H2O)
E	12mM	13mM	14mM	15mM	16mM	neg.(+Rmix)	air
F	12mM	13mM	14mM	15mM	16mM	air	air

Considerations

choosing smaller rx's was needed because of lack of luciferase assay reagent
new luciferase assay reagent AGAIN full of fungi, even after sterile filtering and storage at - 20°
very small pipeting steps between Mg-concentrations: 0,24µl - 0,26µl - 0,28µl - 0,30µl - 0,32µl –> high error quote possible

Results

results have been very inaccurate/ have high errors and do not confirm the findings of the first experiment.
i blame the small rx size and therefore very small pipeting volumes for the errors.

probably another repetition is needed

Maxiprep of HA-GFP-His6-His6 and pBEStluc

(Luisa)

failed: c= 69 ng/µl and 183 ng/µl in 1 ml
Repitiotion of experiment from Tuesday
50 µl reactions in black/transparent 384 well plate over time (2000 ng DNA/reaction)
480/520 nm over time
after 2 hours spectrum 480/500-700 nm with foil and without

plate	lysate	premix	amino acids	DNA	comment
A1 & B1	Bernhard	Koko	ours	-	negative control
A2 & B2	Bernhard	Koko	ours	HA-GFP-His6-His6
A4 & B4	Bernhard	Koko	ours	His10-GFP-Spy
A5	Bernhard	Koko	ours	GFP iGEM Bielefeld
A6	Bernhard	Koko	ours	His10-GFP-Spy
G6 & H6	Bernhard	promega	ours	His10-GFP-Spy
C1 & D1	Koko	Koko	ours	-	negative control
C2 & D2	Koko	Koko	ours	HA-GFP-His6-His6
C4 & D4	Koko	Koko	ours	His10-GFP-Spy
E1 & F1	Promega	Promega	Promega	-	negative control
E2 & F2	Promega	Promega	Promega	HA-GFP-His6-His6
E4 & F4	Promega	Promega	Promega	His10-GFP-Spy
G1 & H1	Bernhard	Promega	Promega	-	negative control
G2 & H2	Bernhard	Promega	Promega	HA-GFP-His6-His6
G4 & H4	Bernhard	Promega	Promega	His10-GFP-Spy
G5	Bernhard	Promega	Promega	Bielefeld GFP
G6 & H6	Bernhard	Promega	Promega	His10-GFP-Spy

Thu, 20.08.2015

Comparison of Koko's and Roth's Kit

(Koko)

Comparing kinetics of GFP expression, with linear and circular templates

in Koko's Mix: 25µl rx's with 1µg DNA each (=optimimum)
in Roth's Mix: 25µl rx's with 0,5µg DNA each (=optimimum)

plate layout

	1	2	3	4
C	Roth negative	Roth mix + 0,5 µg linear	Roth mix + 0,5 µg circular	air
D	Koko mix + 1 µg circular	Koko mix + 1 µg circular	Koko mix + 1 µg linear	Koko negative
…
H	GFP=?	GFP dilution=?

overlaying reactions with 25µl of mineral oil (because Normann said so)
expression in platereader at 37° for 1:30h
fluorescence measurement every 2min
in 384 well plate, transparent, square bottom

added a few measurements after 1:30h because Koko's still looked like it was expressing

RESULTS

Roths don't know their GFP concentrations, so positive control is kinda worthless. Higher curve was green GFP, so probably very high concentrated; other one „1:9“ of unkown start concentration.
Roths Kit is expressing their own linear template in an extreme amount very fast, but nothing else:
circular DNA did not work in Roth's Kit at all
linear DNA did not work in Koko's Kit
very slow expression in Koko's Kit. After 1:30 only at less then 1/10th of usual sample emission values (about 8000-9000 in other experiments)
plasmid DNA is known to express better than linear templates, so why doesn't it work in Roth's Kit at all?
mineral oil still sucks because its harming the reaction, or is at least quenching emission values.

Cellfree expression on NiNTA

3 samples with HA-GFP-His6-His6 DNA (2000 ng in 15 µl) and 3 samples without DNA were spotted on a slide with spotting mask
2 slides were prepared as duplicates
Kokos lysate, premix and own amino acids were used
spotting pattern:

	triplicate	triplicate	triplicate	triplicate
spot	pos (HA-GFP-His6-His6)	neg (no DNA)	GFP (pos control)	bBSA (neg control)
spot	pos (HA-GFP-His6-His6)	neg (no DNA)	GFP (pos control)	bBSA (neg control)
spot	pos (HA-GFP-His6-His6)	neg (no DNA)	GFP (pos control)	bBSA (neg control)

Miniprep of GFP from iGEM Bielefeld

elution in 22 µl nuclease free water
concentration: 389 ng/µl

Maxiprep of HA-GFP-His6-His6 and pBEStluc

2 x 250 ml were incubated over night

Kinetic assay for cellfree expression of luciferase

3 assays were performed by adding luciferin and dilution reagent (25 µl) to 25 µl reactions in a white 96 well plate
measurements were taken every two minutes for 20 minutes (A1/B1 = measurement after 0 minutes of expression, A2/B2 = measurement after 2 minutes of expression, …)
samples were measured for 116 sec
triplicates A, B & C were measured by storing B and C on ice while measuring A (Premix and DNA were added just before measurement)
measurement of C was unreasonably noisy

Tue, 18.08.2015

Western Blot and Dot blot of Expression of today and from first Expression with Vector from Bielefeld
- 25 µl sample was mixed with 7µl 5x SDS-Buffer and heated to 99°C for 15 min
- 25 µl of the samples were pipted on 12,5% SDS Gel
- Gel 1:

^1 ^2 ^3 ^4 ^5 ^6 ^7 ^8 ^9 ^10 ^ 11 ^ 12 ^13 ^ 14 ^ 15^

Marker

E1

E2

E3

f1

F2

F3

G1

G2

G3

H1

H2

J3(mistake)

K1

K2

K3

 
  * Gel2:

Marker

L1

l2

H3(mistake)

I1

I2

I3

J1

J2

L3 (mistake)

PP Bielefeld

PP Bielefeld no DNA

BK Bielefelfd no DNA

BK Bielefeld

F4

* Natural Dot Blor was pipetted following the plate layot

Dot blot with SDS-samples shows the same pipetting mix up like above
run for 1,5 h at 60 mA
Blot for 1,5 h at 0,26A
Blocking in 5% TBS-T with Milkppowder oN @ 4° ( blue box)
Gel was stained over night with Comassie

Expressing all the GFPs

50 µl reactions in black/transparent 384 well plate over time (2000 ng DNA/reaction)
480/520 nm over time
after 2 hours spectrum 480/500-700 nm with foil and without

plate	lysate	premix	amino acids	DNA	comment
E1 & F1	Bernhard	Koko	ours	-	negative control
E2 & F2	Bernhard	Koko	ours	HA-GFP-His6-His6
E3 & F3	Bernhard	Koko	ours	His10-GFP-Spy
E4 & F4	Bernhard	Koko	ours	GFP iGEM Bielefeld
G1 & H1	Koko	Koko	ours	-	negative control
G2 & H2	Koko	Koko	ours	HA-GFP-His6-His6
G3 & H3	Koko	Koko	ours	His10-GFP-Spy
G4 & H4	Bernhard	Promega	Promega	GFP iGEM Bielefeld
I1 & J1	Promega	Promega	Promega	-	negative control
I2 & J2	Promega	Promega	Promega	HA-GFP-His6-His6
I3 & J3	Promega	Promega	Promega	His10-GFP-Spy
I4 & J4	-	-	-	-	air
K1 & L1	Bernhard	Promega	Promega	-	negative control
K2 & L2	Bernhard	Promega	Promega	HA-GFP-His6-His6
K3 & L3	Bernhard	Promega	Promega	His10-GFP-Spy
K4 & L4	-	-	-	-	air

Mon, 17.08.2015

Expression of GFP DNA sample from iGEM Bielefeld (BK, PP)

tube reactions are measured in plate reader at 488nm/500nm
a western blot with GFP antibody will be performed

Expression of luciferase in platereader (over time)

Sun, 16.08.2015

Western Blot of expression of pQE-HA-GFP-his-his (Koko)

gel loading pattern: (12% sds-page, 0,? A, 1:30h)

1	2	3	4	5	6	7	8	9	10
ladder (ProSieve 4-300kDA)	25µl rx w/ DNA	25µl rx w/ DNA	25µl rx, no DNA	25µl rx, no DNA	GFPhis 500µg/ml	GFPhis 100µg/ml	GFPhis 10µg/ml	GFPhis 1µg/ml	buffer

blotting onto PVDF membrane: (0,25A, 1h)
checking for correct transfer using ponceau staining (was correct)

* antibody staining:

primary (Rockland anti GFP (goat)) 1:2000, at 4°C o/n
secondary (Rockland anti Goat HRP (rabbit)) 1:10.000, RT, 1:30h

blotting and washing procedure after towbin et al. protocol

Results:

clear bands in Rx w/ DNA at height of GFP-controls at the right size.
some smaller extra protein visible above GFP band

—> incomplete translational product containing epitope recognized by AB?

Dot Blot of HA-GFP-His6-His6 to test varying plates and coverage over time

(both)

3 X washing with TBS-T
1 AB anti GFP from Rockland (1:2000) in 2% BSA
3x washing with TBS-T
2nd AB anti goat

plate was loaded upside down (A = P), blot has to be evaluated the other way around

Expression of GFP DNA sample from iGEM Bielefeld (BK, PP)

(Luisa)

on plate	reaction size	performed in	covered with	DNA	premix	lysate	comment
A 4-6	50 µl	tube, measured in 384 well plate	-	none	Koko	Bernhard	negative control
B 4-6	50 µl	tube, measured in 384 well plate	-	Bielefeld	Koko	Bernhard	sample
C 4-6	50 µl	tube, measured in 384 well plate	-	none	Promega	Promega	negative control
D 4-6	50 µl	tube, measured in 384 well plate	-	Bielefeld	Promega	Promega	sample
J 4-6	10 µl, 1:32	tube, measured in 384 well plate	-	none	none	none	expressed GFP, positive control
K 4-6	10 µl, 1:64	tube, measured in 384 well plate	-	none	none	none	expressed GFP, positive control
L 4-6	10 µl, 1:128	tube, measured in 384 well plate	-	none	none	none	expressed GFP, positive control

reactions are performed over 2 hrs at 37°C in tubes
incubated for folding at 4°C over night
tube reactions are measured in plate reader at 488nm/500nm

PCRs for more HA-GFP-His6-His6 with Cy3 and amino label

(Luisa)

PCRs will be performed in a 100 µl reaction without DMSO in GC buffer
forward primer is Cy3 labeled, reverse primer has amino label

program HA-GFP-His6-His6

Temperature °C	duration
98	5 min
98	20 sec
69	20 sec
72	60 sec
72	5
4	infinite

Component	amount [µl]
buffer - Phusion GC	20
dNTPs	5
Primer forward (Cy5 labeled)	5
Primer reverse (amino labeled)	4
Phusion Polymerase	1
Template (pre-purified) <250 ng	2
DMSO	0
H2O to 100µl each	63

trying another 100 µl PCR was just stupid!

Repetition PCRs for more HA-GFP-His6-His6 with Cy3 and amino label

(Luisa)

PCRs was performed in 2 x 50 µl reaction without DMSO in GC buffer
forward primer is Cy3 labeled, reverse primer has amino label

program HA-GFP-His6-His6

Temperature °C	duration
98	5 min
98	20 sec
69	20 sec
72	60 sec
72	5
4	infinite

Component	amount [µl]
buffer - Phusion GC	10
dNTPs	2.5
Primer forward (Cy5 labeled)	2.5
Primer reverse (amino labeled)	2
Phusion Polymerase	0.5
Template (pre-purified) <250 ng	1
DMSO	0
H2O to 100µl each	31.5

→ per tube

purify one tube for iRIf, hand over unpurified PCR product of other tube
PCR worked, 2nd tube: 44 ng/µl in 17 µl nuclease free water

Sat, 15.08.2015

TO DO (Koko)

~~WesternBlot of the //HA-GFP-His6-His6// from Wed, 12.08~~

make moar plasmid! (pQEHA-GFP-His6-His6)

Fri, 14.08.2015

Using the //HA-GFP-His6-His6// from Wed, 12.08 for surface immobilization (Koko)

collaboration with SurRIf subgroup.

spotting onto Ni-NTA surfaces, by hand, using a stencil for uniformity of the spots (made of PDMS)

spotting pattern:

spot	component	concentration
1	Reaction with HA-GFP-his-his	unknown
2	Reaction with HA-GFP-his-his	unknown
3	Reaction with HA-GFP-his-his	unknown
4	Reaction w/o DNA	unknown
5	Reaction w/o DNA	unknown
6	Reaction w/o DNA	unknown
7	hisGFP lysate	~1 mg/ml
8	hisGFP lysate	~1 mg/ml
9	untagged GFP	~1 mg/ml
10	bBSA	200 µg/ml
11	Max GFP	0,5mg/ml

all spots contain 2µl and have been incubated on the slide over night at 4° in a damp environment (wet towels) to keep them from evaporating.
afterwards, the PDMS spotting mask is taken off
the glass slide is N2-dried (using a wafer-gun)
slide is put onto PDMS-flowchamber, protein-side facing inwards, and softly pressed on.
slide and flowchamber are then being put into the iRIf-device and connected to the microfluidics.

Flush protocol:

Reagent	#	Flowrate	Priming time	Concentration
Buffer	1	60	780	1x
BSA	2	60	780	10 mg/ml
Buffer	3	60	600	1x
anti HA	4	40	450	5 ug/ml
Buffer	5	60	300	1x
anti GFP	6	40	450	3 ug/ml
Buffer	7	60	600	1x
Strep Cy5	8	40	450	5 ug/ml
Buffer	9	60	600	1x

Results:

Roi selection Exp. 46b: Spot 11 couldnt be selected due to air bubble on it

Binding curves of Exp. 46b for all spots

Binding curves of Exp. 46b only for spots 1-6

Spot 11 couldnt be evaluated due to air on it. Spot 1-3 show a-HA binding (weak but clear) and good a-GFP binding → successful cellfree expression.
No strepcy5 binding can be detected: Tube to flowchamber pulled off just before that step….

Expression of HA-GFP-His6-His6 to test varying plates and coverage over time

(Luisa)

on plate	reaction size	performed in	covered with	DNA	premix	lysate	comment
A 1-3	15 µl	384 well plate	foil	none	Koko	Bernhard	negative control
B 1-3	15 µl	384 well plate	foil	HA-GFP-His6-His6	Koko	Bernhard	sample
C 1-3	15 µl, 1:8	384 well plate	foil	none	none	none	expressed GFP, positive control
D 1-3	15 µl	384 well plate	mineral oil	none	Koko	Bernhard	negative control
E 1-3	15 µl	384 well plate	mineral oil	HA-GFP-His6-His6	Koko	Bernhard	sample
F 1-3	15 µl 1:8	384 well plate	mineral oil	none	none	none	expressed GFP, positive control
G 1-3	15 µl	tube, measured in plate	nothing, foil	none	Koko	Bernhard	negative control
H 1-3	15 µl	tube, measured in plate	nothing, foil	HA-GFP-His6-His6	Koko	Bernhard	sample
I 1-3	15 µl, 1:8	tube, measured in plate	nothing, foil	none	none	none	expressed GFP, positive control
J 1-3	15 µl, 1:16	tube, measured in plate	nothing, foil	none	none	none	expressed GFP, positive control
K 1-3	15 µl, 1:32	tube, measured in plate	nothing, foil	none	none	none	expressed GFP, positive control
L 1-3	15 µl, 1:64	tube, measured in plate	nothing, foil	none	none	none	expressed GFP, positive control

reactions are performed over 2 hrs at 37°C
tube reactions are measured in plate reader after reaction and covered with foil after first measurement

Thu, 13.08.2015

purification of HA-GFP-His6-His6 with Cy3 and amino label (Luisa)
- concentration: 28 ng/µl
- tubes were covered with aluminium foil to avoid bleaching and handed to surface chemistry

Repetition of variation of DNA-concentration, 1-5µg (Koko)

Due to criticism in the meeting for lacking duplicates or triplicates, the experiment was repeated using biological triplicates for each concentration. (=3 own reactions (from mastermix) for each conc., including MgOAc-feeding)
plate layout:

	5	6	7	8	9	10	11	12
A	1	2	3	1	2	3	1	2
B
C
D
E
F

^ ^ 5 ^ 6 ^ 7 ^ 8 ^ 9 ^ 10 ^ 11 ^ 12 ^

A	1 µg 1	1 µg 2	1 µg 3	2 µg 1	2 µg 2	2 µg 3	3 µg 1	3 µg 2
B	3 µg 3	4 µg 1	4 µg 2	4 µg 3	5 µg 1	5 µg 2	5 µg 3	neg 1
C	neg 2	neg 3
D	1 µg 1	1 µg 2	1 µg 3	2 µg 1	2 µg 2	2 µg 3	3 µg 1	3 µg 2
E
F	neg 2	neg 3

Results

Wed, 12.08.2015

(Koko)

Expression of HA-GFP-His6-His6

50µl reaction of complete Koko-system
using the evaluated best-working DNA concentration of 2 µg (see 08.08.)
using the evaluated best-working MgOAc-concentration of 14mM (see 10.08)
2h, 37°C, 0rpm

planned to use for:

~~spotting the finished expression onto Ni-NTA slides in collaboration with SurChem subgroup, to check for working immobilization of the tagged GFP~~

WesternBlot to confirm correct expression

Repetition of MgOAc-variation in Bernhard-Koko system from Mon, 10.08.

this time using the right concentrations, otherwise exactly the same

Results:

system completely dead, no expression
–> Bernhard mix does not, as promised, work best between 15 and 18mM.
other explanation: our stock of MgOAc is unknowingly slightly higher concentrated than we think it is

PCRs of HA-GFP-His6-His6 with Cy3 and amino label

(Luisa)

PCRs will be performed in a 50 µl reaction without DMSO in GC buffer
forward primer is Cy3 labeled, reverse primer has amino label

program HA-GFP-His6-His6

Temperature °C	duration
98	5 min
98	20 sec
69	20 sec
72	60 sec
72	5
4	infinite

Component	amount [µl]
buffer - Phusion GC	10
dNTPs	2.5
Primer forward (Cy5 labeled)	2.5
Primer reverse (amino labeled)	2
Phusion Polymerase	0.5
Template (pre-purified) <250 ng	1
DMSO	0
H2O to 100µl each	31.5

Tue, 11.08.2015

Continuation of Western and Dot Blot of Expression of pIG15_105 from yesterday (Sabi)
- 3 x washing with TBS-T
- first antibody: anti-tYFP (Evrogen) (1:5000)
- 3 x washing with TBS-T
- sec. antibody: anti-rabbit 1:20000 (Promega)
- 3x washing with TBS-T
- Signal at the right hight around 61 kDa (ca 27kDa tYFP + 34 kDa Halo)

Mon, 10.08.2015

Variation of start-concentration of MgOAc (Koko)
- in both Bernhard+Koko and complete Koko for further improvement of reaction conditions
- Bernhard once said his system was optimal in the range of 15-18mM.
- no further feeding of the mix.
- 2h, 37°C, 0rpm
- 4µg of pBESTluc DNA to compare expression via Luciferase assay later on

a terrible mistake was made while pipeting the B-K reaction (using 1M stock of MgOAc instead of 10µM to adjust molarity)

first one was not (falsely) adjusted (= 11mM)–> visible maximum
–> will be repeated (see 12.08.)

Results:

Dot Blot and Western Blot of Expressionfrom today (105): (Sabi)
- before measuring in platereader: 10 µl of reaction were passed on to SurChem
- 15µl were used for measurement in platereader
- the remaining 25 µl were mixed with 7 µl 5X SDS sample buffer and heated too 99°C for 15 min
- 2µl of the samples were spotted for a Dot Blot on activated PVDF
  - After measurement in the platereader 2 µl of the reaction mix that were spotted as well (without SDS)
- for the Western Blot 25µl sample were used
  - Pippetting sheme: Marker-no DnA-K/K-K/K+feed-B/K-B/K+feed
    - gel run for 1,5 h at 0,03 A but looked slightly strange –> SDS sample buffer might be invested with foreign proteins
    - in the biginning gel rumn very slow only on centimeter in an hour. Probably due to the missing second balance glass plate. Glass plate was attached and the run started again
    - blotting at 0,14A for 1,5 h with the small blotting apperature
      - whole marker was on the membrane and a little bit on the Whatman behind –> no further increase of Ampere
- blocking in TBS-T with 5% milkpowder oN at 4°C

Continuation of Dot and Western blot from yesterday (104)
3x washing with TBS-T
first anti-body: anti-YFP (1:5000)
3x Washing with TBS-T
sec. Antibody: anti Rabbit (1:20000)
3 washing with TBS-T
- clear spots with KK+ /KK- –> However in the platereader B performed better
- only natural spotted expression mix shows clear signal –> perhaps due to differnt amount of proteins, as indicated by the ponceau staining
- two bands at the height of tYFP

expression of pIG15_105 (Prep) (Luisa)

cellfree expression of pIG15_105 (His-tYFP-Spy) by using 5 µg of DNA with a mix of various lysates, amino acids and premixes
selfmade system of Koko and Bernhard lysate with premix of Koko were fed with 0.5 µl Mg(OAc)2 every 20 minutes
fluorescence measurement was performed at an excitation of 488 nm

K = Koko

B = Bernhard

Lysate	Premix	Amino Acids	Feed?	comment
K	K	ours	Mg(OAc)2	-
K	K	ours	H2O	-
B	K	ours	Mg(OAc)2	-
B	K	ours	H2O	-
K	K	ours	H2O	negative control

Sun, 09.08.2015

Continuation of Dot Blot of GFP Expression from yesterday (Sabi)
- 3 X washing of membrane with TBS-T
- exposure for 4 min and 8 min

Dot Blot of Expression YFP from yesterday: (Sabi)

Dot Blot and Western Blot of Expressionfrom today: (Sabi)
- before measuring in platereader: 10 µl of reaction were passed on to SurChem
- 15µl were used for measurement in platereader
- the remaining 25 µl were mixed with 7 µl 5X SDS sample buffer and heated too 99°C for 15 min
- 2µl of the samples were spotted for a Dot Blot on activated PVDF
  - After measurement in the platereader 2 µl of the reaction mix that were spotted as well (without SDS)
- for the Western Blot 25µl samples were used
  - Pippetting scheme: Marker-no DnA-K/K-K/K+feed-B/K-B/K+feed
    - gel run for 2 h at 0,03 A but looked slightly strange –> SDS sample buffer might be contaminated invested with other proteins
    - blotting at 0,13A for 1,5 h

loose contact at big blotting apparatus

together with teh Dot Blot the membranes were blocked oN in 5% Milkpowder TBS-T

repeat of yesterdays expression with pIG15_104 by using the intended amount of DNA (5 µg) (Luisa)

cellfree expression of pIG15_104 (His-tYFP-Spy) by using 5 µg of DNA with a mix of various lysates, amino acids and premixes
selfmade system of Koko and Bernhard lysate with premix of Koko were fed with 0.5 µl Mg(OAc)2 every 20 minutes
fluorescence measurement was performed at an excitation of 488 nm

K = Koko

B = Bernhard

Lysate	Premix	Amino Acids	Feed?	comment
K	K	ours	Mg(OAc)2	-
K	K	ours	H2O	-
B	K	ours	Mg(OAc)2	-
B	K	ours	H2O	-
K	K	ours	H2O	negative control

Sat, 08.08.2015

Variation of DNA-concentration from 1-5µg

5 expressions and a negative control in 50µl size were carried out in parallel:
- Mastermix for 5 rx:

component	amount from stock in µl
S30 Lysate	112,5
HEPES-KOH ph 7,5	13,75
DTT	4,25
ATP pH7	3
CTP	2
GTP	2
UTP	2
Creat.Phos	20
CreatineKin	0,875
PEG4k	5
cAMP	1,625
folinic acid	8,75
tRNA	5,7
pot.glutamat	18,5
MgOAc	2,75
Aminos	9,375

The Premix for 5 rx w/o lysate adds up to 99,575µl = 19,915µl per 50µl reaction.
The negative control consists of 22,5µl lysate + 27,5µl water, no DNA.
Reaction carried out as usual: 2h, 37°C, 0rpm
Reactions were feeded 0,5µl of 10µM MgOAc every 20min.

DNA variation:

Expression of pBESTluc plasmid (c=3151,4ng/µl)

DNA	amount from stock in µl
1µg	0,31
2µg	0,63
3µg	0,95
4µg	1,27
5µg	1,58
0µg	1,00 (H2O)

6,01-7,28 µl of water ad 50µl

Luciferase Assay to compare expression:

20µl of reaction+ 20µl of dilution agent for each reaction
in 96well plate, white, flatbottom
adding 50µl of luciferase assay reagent (=luciferin+cofactors) to each well right before measurement
full spectrum scan (300-700nm) in microplate reader

Results

highest expression for DNA=2µg
negative control showed signal higher than 1µg, which suggests errors in pipeting or mix up of data.
Experiment was repeated with triplicates on 13.08.15

Dot Blot of GFP of last Expression from yesterday (Sabi)
- on the activated PVDF membrane the 7 samples and a GFP positiv control were spotted
- The membrane was blocked for 1 hour in 5%milkpowder in TBS-T
- 3 x washing with TBS-T
- 2 h incubation with anti GFP (1:2000) (mouse)
- 3 x washing with TBS-T
- incubation oN at 4°C with anti mouse ab

measurement of the GFP Expression from 7.8.2015 (Sabi)
- Excitation 488 –> Emission 509-700 spectral scanning
- white plates
- remeasurement with black plates and see-through plates
  - no big difference between those kinds of plates. however white plaes increase the signal and gives a distorted picture of reality.
    - for example were the negative controls over 20000 relative fluorecence units

never use white plate for fluorecence

expression of pIG15_104 (Prep) (Luisa)

cellfree expression of pIG15_104 (His-tYFP-Spy) by using 0.5 µg of DNA with a mix of various lysates, amino acids and premixes
selfmade system of Koko and Bernhard lysate with premix of Koko were fed with 0.5 µl Mg(OAc)2 every 20 minutes
after expression the plate was measured covered with foil and without foil to test wether an expression in the platereader without evaporation of the reaction would be possible
fluorescence measurement was performed at an excitation of 488 nm

K = Koko

B = Bernhard

Lysate	Premix	Amino Acids	Feed?	comment
K	K	ours	Mg(OAc)2	-
K	K	ours	H2O	-
B	K	ours	Mg(OAc)2	-
B	K	ours	H2O	-
K	K	ours	H2O	negative control

Fri, 07.08.2015

expression of HA-GFP-His6-His6 (Prep) (Koko)

cellfree expression of HA-GFP-His6-His6 by using 3,25 µg of DNA with a mix of various lysates, amino acids and premixes
selfmade system of Koko and Bernhard lysate with premix of Koko were fed with 0.5 µl Mg(OAc)2 every 20 minutes
fluorescence measurement was performed at an excitation of 488 nm

K = Koko

B = Bernhard

P = Promega S30 for linear templates

Lysate	Premix	Amino Acids	Feed?	comment
K	K	ours	Mg(OAc)2	-
K	K	ours	no	-
B	K	ours	Mg(OAc)2	-
B	K	ours	no	-
B	P	P	no	-
P	P	P	no	positive control
K	H2O	H2O	no	negative control

short: BK = lysate Bernhard, Premix Koko
the negative control contains KK and no DNA

repetition of gel control of pIG15_104 and 105 by loading 20 µl on a gel (Luisa)

Purification of PCR product of aminylated linear 104 and 105 (Sabi)
- elution with 5 µl water
Purfication of PCR product of linear 104 and 105
- Elution with 12,5 µl water

Miniprep of pIG15_104, pIG15_105 and PQE HA-GFP-His6-His6
- Elution with 25µl

== PCR for linear template of pIG15_1104 (His10-C.tetani-Spy) == (Luisa) * length 1371 bp * primers oIG15_s001 and pIG15_s003 * program ^ Temperature °C ^ duration ^ |98|5 min | |98|20 sec | |52 °C |20 sec | |72 |70 sec | |72 |5 | |4 |infinite | * 1 reaction, 50 µl ^ Component ^ amount [µl] ^ |buffer - Phusion HF|10 | |dNTPs| 2.5 | |Primer forward |4.4 | |Primer reverse |4.4 | |Phusion Polymerase |0.5 | |Template (pre-purified) <250 ng |1 | |DMSO | 1.25 | |H2O to 100µl each | 26.75 | (not yet performed)

Thu, 06.08.2015

PCR for high yield template of pIG15_105 and pIG15_a105 (Amino-His10-tYFP-Halo)

was performed with 50 µl aliquots of a 500 µl master mix using 1.25 µl DMSO and 2.5 µl dNTPs
for aminylated PCR product 1 x 50 µl reaction was tested
increased elongation time to 120 sec

program pIG15_105

Temperature °C	duration
98	5 min
98	20 sec
52 °C	20 sec
72	120 sec
72	5
4	infinite

Master Mix

Component	amount [µl]
buffer - Phusion HF	110
dNTPs	27.5
Primer forward	44
Primer reverse	44
Phusion Polymerase	0.5 per tube
Template (pre-purified) <250 ng	11
DMSO	13.75
H2O to 100µl each	294.25

for pIG15_a105

Component	amount [µl]
buffer - Phusion HF	10
dNTPs	2.5
Primer forward	4
Primer reverse	4
Phusion Polymerase	0.5
Template (pre-purified) <250 ng	1
DMSO	1.25
H2O to 100µl each	26.75

PCR for linear template of pIG15_104 (His10-tYFP-Spy), pIG15_a104 (Amino-His10-tYFP-Spy)

for regular pIG15_104 the PCR was performed in 10 x 50 µl reactions with an increased elongation time
for aminylated PCR product 1 x 50 µl reaction was tested

program pIG15_104 and pIG15_a104

Temperature °C	duration
98	5 min
98	20 sec
52 °C	20 sec
72	60 sec
72	5
4	infinite

→ elongation time was increased to 60 seconds

Master Mix pIG15_104

Component	amount [µl]
buffer - Phusion HF	110
dNTPs	27.5
Primer forward	44
Primer reverse	44
Phusion Polymerase	1 per tube
Template (pre-purified) <250 ng	11
DMSO	13.75
H2O to 100µl each	294.25

for pIG15_a104

Component	amount [µl]
buffer - Phusion HF	10
dNTPs	2.5
Primer forward	4
Primer reverse	4
Phusion Polymerase	0.5
Template (pre-purified) <250 ng	1
DMSO	1.25
H2O to 100µl each	26.75

Luciferase Dilution Reagent

adapted from Promega S30 Kit for linear templates

concentration	component	comment
25 mM	Tris-Phosphate (pH 7.8)
2 mM	DTT
2 mM	edta	in original protocol: 1,2-diaminocyclohexane- N,N,N',N',-tetraacetic acid
10 %	glycerol
1 %	Triton X-100
1 mg/ml	BSA

Wed, 05.08.2015

western blot and dot blot started yesterday 4.8.15:

3x washing with TBS-T
1nd Antibody:
anti GFP Mouse 1:3000 in TBS-T
anti-Luc mouse 1:500 in TBS-T
3x washing with TBS-T
2nd AB: anti Mouse 1:20000
3X washing with TBS-T

Comassie of Western Blot

next time gel should be run at increased Ampère. In the applied running time, the bands didn't separate well
perhaps a change of gel concentration would be beneficial as well

Ponceau staining of western membranes

Dot blot shows some clear protein spots
not a lot of protein is visible in the lines of the western blot
some of the samples weren't visible at all –> repeat of western necessary → preparation of bigger samples necessary

Western blot(transmitted light picture; EcL-Picture[3 min]; picture of arrangement taken with camera )

Anti-GFP shows bands in the lane of B+P –> however bands are at 15kDa it should be at 27kDa

Dot Blot-GFP (transmitted light picture; EcL-Picture[3 min]; picture of arrangement taken with camera)

Dot blot LUC (transmitted light picture; EcL-Picture[3 min]; picture of arrangement taken with camera)

PCR for linear template of HA-GFP-His6-His6

(Luisa)

test HA-GFP-His6-His6 PCR in 6 x 25 µl reactions

Sample	buffer	DMSO [µl]	H2O [µl]
1	Phusion HF	0	12.875
2	Phusion HF	0.5	13.375
3	Phusion HF	1	13.875
4	Phusion GC	0	12.875
5	Phusion GC	0.5	13.375
6	Phusion GC	1	13.875

program HA-GFP-His6-His6

Temperature °C	duration
98	5 min
98	20 sec
69	20 sec
72	60 sec
72	5
4	infinite

Master Mix

Component	amount [µl]
buffer - Phusion HF or GC	15.1 each
dNTPs	-
Primer forward	6.04 each
Primer reverse	6.04 each
Phusion Polymerase	0.755 each
Template (pre-purified) <250 ng	1.51 each
DMSO	-
H2O to 100µl each	-

elongation time was increased to 1 minute
10 µl of PCR product were tested on a gel
from now on HA-GFP-His6-His6 PCRs should be performed using no DMSO and Phusion GC buffer

PCR for high yield template of HA-GFP-His6-His6

(Luisa)

the PCR will be performed in 10 x 50 µl reactions, while loading 10 µl on a gel as control

program HA-GFP-His6-His6

Temperature °C	duration
98	5 min
98	20 sec
69	20 sec
72	60 sec
72	5
4	infinite

Master Mix

Component	amount [µl]
buffer - Phusion GC	110
dNTPs	27.5
Primer forward	44
Primer reverse	44
Phusion Polymerase	5.5
Template (pre-purified) <250 ng	11
DMSO	-
H2O to 100µl each	294.25

the bands run a little too high. re-check!

Tue, 04.08.2015

cellfree expression of digested GFP and luciferase

(both)

compare expression of GFP and Luciferase in:
- Bernhard lysate + own amino acids + own premix + single components (PEP, ATP, GTP, CTP, UTP, tRNA, CoA) + 2000 ng GFP and 1500 ng luciferase DNA in DECP water
- Bernhard lysate + promega amino acids + promega premix + 2000 ng GFP and 1500 ng luciferase DNA in DECP water
- promega lysate + promega amino acids + promega premix + 2000 ng GFP and 1500 ng luciferase DNA in DECP water
- negative controls:
  - own lysate following EMBL protocol (Koko) + own amino acids + own premix + DECP water, no DNA
  - promega lysate + promega amino acids + promega premix + DECP water, no DNA

reaction was performed at 30 °C, 300 rpm for 2h in the thermomixer
afterwards samples were kept at 4°C

PCR for linear template of pIG15_105 and HA-GFP-His6-His6

(Luisa)

test HA-GFP-His6-His6 PCR from yesterday by loading 20 µl to a gel
gel control of pIG15_105 PCR from yesterday

purification of pIG15_105 PCR (Sabi):
- elution in: 12,5 ul on 2 columns
- concentration: 950 ng/µl

Western Blot of expression of pIG15_105 and HA-GFP-His6-His6 from today:
- big 12 % SDS Gel with Collecting gel was cast ( next time use of red comb!)
- 24 ul of the GFP samples and negative controls were mixed with 6ul SDS 5X running buffer and heated to 95 °C for 10 min
- for Luc samples: due to not enough sample after back and forth pipetting on the plate only 20 µl sample were used
- 15 ul of the samples were put onto the blot. However pipetting was quite difficult due to solid components in the sample and inhomogenity ( perhaps next time the use of more sample is necessary. Also shering of samples with thin pipette could be tried)
- gel run for ca 2 h at 0,02 A
- Blotting of Gel for 1,5 h at 0,12 A
- Gel was stained with Comassie oN
- blot was blocked oN at 4°C( together with the dot blots) in 5% milk powder TBS-T

Dot Blot of expression of pIG15_105 and HA-GFP-His6-His6 from today (Sabi):
- pipetting scheme : marked with a pen on the blot
1µl of the samples were pipetted onto the methanol-activated PVDF membrane
- Membrane was still slightly wet or rewetted for the drops to be able to seep in
  - with SDS buffer prepared samples were spotted onto the membrane
  - additionally non treated samples were spotted onto the membrane
    - for Luciferase: samples that were diluted and mixed with reaction reagent from the plate reader were used
- Membrane was blocked with TBS-T with 5% milk powder oN

Mon, 03.08.2015

Dot blot of Expression of pIG15_105 and HA-GFP-His6-His6 from yesterday: (Sabi)
- pipetting scheme

2ul of the samples were pipetted onto the methanol-activated PVDF membrane
- Membrane still has to be slightly wet or drops aren`t able to seep into the membrane
- Membrane was blocked with TBS-T with 5% milk powder for 1 h (and specific blocking buffer for His-Conjugate)
- 3 X washing with TBS-T
- First AB for 2 h in TBS-T:
  - anti-tYFP 1:5000 in TBS-T Rabbit polyclonal
  - anti LUC 1:500 in TBS-T Mouse monoclonal
  - anti His-Conjugate (Qiagen) incubated on membrane until the other blots were ready for final washing steps
- 2nd AB for 1 h in TBS-T:
  - anti_Mouse (1:20000) for Luc
  - anti- Rabbit (1:20000) for tYFP
- 3 X washing with TBS-T
- Blots in Reader: YFP ( left up); Luc (right up); His (left down)

PCRs of pIG15_105 and HA-GFP-His6-His6

(Luisa)

PCRs will be performed in 6 x 50 µl reactions with varying dNTP and DMSO concentrations

Sample	dNTPs [µl]	DMSO [µl]
1	2.5	1.25
2	2.5	1.5
3	2.5	1.75
4	3	1.25
5	4	1.25
6	5	1.25

program HA-GFP-His6-His6

Temperature °C	duration
98	5 min
98	20 sec
69	20 sec
72	30 sec
72	5
4	infinite

program pIG15_105

Temperature °C	duration
98	5 min
98	20 sec
52 °C	20 sec
72	70 sec
72	5
4	infinite

Master Mix

Component	amount [µl]
buffer - Phusion HF	66 each
dNTPs	-
Primer forward	26.4 each
Primer reverse	26.4 each
Phusion Polymerase	0.5 per tube
Template (pre-purified) <250 ng	6.6 each
DMSO	-
H2O to 100µl each	176.55 each

PCR for HA-GFP-His6-His6 did not work because of problems with the thermocycler
- PCR was repeated with protocol above, with addition of a 7th sample containing 1.25 µl DMSO, 2.5 µl dNTPs and Phusion GC buffer instead of Phusion HF buffer

final PCR for pIG15_105 was performed with 50 µl aliquots of a 500 µl master mix using 1.25 µl DMSO and 2.5 µl dNTPs.

program pIG15_105

Temperature °C	duration
98	5 min
98	20 sec
52 °C	20 sec
72	70 sec
72	5
4	over night

Master Mix

Component	amount [µl]
buffer - Phusion HF	110
dNTPs	27.5
Primer forward	44
Primer reverse	44
Phusion Polymerase	0.5 per tube
Template (pre-purified) <250 ng	11
DMSO	13.75
H2O to 100µl each	294.25

Sun, 02.08.2015

(Sabi)

Purification and Concentration of lin PQE-HA-GFP-2(HIS)6 with the Qiagen Kit
- 0,5 ml were purified on two columns and eluted with a 10ul
- only 100ng/ul
- improvement of PCR protocol necessary as the controlgel also shows some degradation

Cellfree Expression of lin PIG15_104 and Luciferse

Reactions were performed in tubes
50 ul reactions at 30°C for 2h
part of the reactions were feeded every 20 min with a 0,5 ul Mg solution
after completion of reaction samples were transfered to a white 96 well plate for measurement of luminescence and fluorescence measurement

the single parts of the reaction mix were useed in relation to the promega mix

Amino Acids	6,00
S30 Premix	20,00
S30 Extrakt	15,00
Template	x (1300ng)
H20	a.D. 50 ul

In several of the reactions parts of the kit were exchange for our own mixes to test the quality of the single components

Pipetting sheme

	8	9	10	11	12
A	Promega Kit (luc 1:2)	Promega Kit with our Premix (Luc 1:2)	Promega Kit with our Aminoacids (Luc 1:2)	Promega Kit with our Berni Lyaste(Luc 1:2)	Bernhard reaction (Luc 1:2)
B
C		Promega lin 104	Promega lin 104 +feed	Bernhard lin 104	Bernhard lin 104 +feed
D		Promega with Berni Lysat lin 104	Promega with Berni Lysat lin 104 +feed lin 104	Promega neg.

* Analysis:

feeding didn`t change the outcome
expression of luciferase probably didn`t work–> signal usually at 580nm
- no expression anywhere –> problems with DNA

repetition of experiment necessary

Sat, 01.08.2015

(Sabi)

Purification and concentration of linIG15_104 with Qiagen Kit
- 104_: 750ng/ul
- pOE-Ha-GFP-2xHis: 60ng/l
- repeat of PCR from tuesday with an 1:10 Dilution of the purified product

Team:Freiburg/Labjournals/Cellfree/August

Labjournal Cellfree August

Thu, 27.08.2015

Wed, 26.08.2015

Expression of HA-GFP-His6-His6 on NiNTA slide

Tue, 25.08.2015

Expression of HA-GFP-His6-His6 on NiNTA slide

Mon, 24.08.2015

Expression of pIG_1104 (tetanus)

Sun, 23.08.2015

Concentration of maxi prep of HA-GFP-His6-His6 and pBEStluc

Ponceau staining of expression of various GFPs

Sat, 22.08.2015

Western blot of expression of various GFPs

Fri, 21.08.2015

Repetition of variation of MgOAc start concentration in triplicates

Maxiprep of HA-GFP-His6-His6 and pBEStluc

Thu, 20.08.2015

Comparison of Koko's and Roth's Kit

Cellfree expression on NiNTA

Miniprep of GFP from iGEM Bielefeld

Maxiprep of HA-GFP-His6-His6 and pBEStluc

Kinetic assay for cellfree expression of luciferase

Tue, 18.08.2015

Expressing all the GFPs

Mon, 17.08.2015

Expression of GFP DNA sample from iGEM Bielefeld (BK, PP)

Expression of luciferase in platereader (over time)

Sun, 16.08.2015

Western Blot of expression of pQE-HA-GFP-his-his (Koko)

Dot Blot of HA-GFP-His6-His6 to test varying plates and coverage over time

Expression of GFP DNA sample from iGEM Bielefeld (BK, PP)

PCRs for more HA-GFP-His6-His6 with Cy3 and amino label

Repetition PCRs for more HA-GFP-His6-His6 with Cy3 and amino label

Sat, 15.08.2015

Fri, 14.08.2015

Using the //HA-GFP-His6-His6// from Wed, 12.08 for surface immobilization (Koko)

Expression of HA-GFP-His6-His6 to test varying plates and coverage over time

Thu, 13.08.2015

Repetition of variation of DNA-concentration, 1-5µg (Koko)

Wed, 12.08.2015

Expression of HA-GFP-His6-His6

Repetition of MgOAc-variation in Bernhard-Koko system from Mon, 10.08.

PCRs of HA-GFP-His6-His6 with Cy3 and amino label

Tue, 11.08.2015

Mon, 10.08.2015

Sun, 09.08.2015

Sat, 08.08.2015

Variation of DNA-concentration from 1-5µg

Fri, 07.08.2015

Thu, 06.08.2015

PCR for high yield template of pIG15_105 and pIG15_a105 (Amino-His10-tYFP-Halo)

PCR for linear template of pIG15_104 (His10-tYFP-Spy), pIG15_a104 (Amino-His10-tYFP-Spy)

Luciferase Dilution Reagent

Wed, 05.08.2015

PCR for linear template of HA-GFP-His6-His6

PCR for high yield template of HA-GFP-His6-His6

Tue, 04.08.2015

cellfree expression of digested GFP and luciferase

PCR for linear template of pIG15_105 and HA-GFP-His6-His6

Mon, 03.08.2015

PCRs of pIG15_105 and HA-GFP-His6-His6

Sun, 02.08.2015

Sat, 01.08.2015