Team:Goettingen/NoteBook w6

Mini Plasmid Preparation

-          3x 4 ml ON culture of plasmid part 7 clone 1

-          1x 4 ml ON culture of RBS-GFP-Terminator clone 12

-          With Macherey-Nagel kit

-          Elution in 30 µl HPLC water

-          Pooling of 3 samples of part 7

-          NanoDrop concentration measurement:

 

	Concentration (ng/µl)	A260nm/A280nm	A260nm/A230nm
Part 7 plasmid	179.3	1.79	1.38
Plasmid RBS-GFP-Terminator C12	200.8	1.87	1.83

 

è Samples stored at -20 °C (pink rag)

Test Digest of Clone 12 RBS-GFP-Terminator (see gel 11.07.) with EcoRI

 

Test Digest pipette as follows:

 

1,5µl DNA (~300ng)

0.1µl EcoRI FD

1.5µl FD Buffer

11µl H20

 

àincubation at 37°C for 45 minutes

àAnalysis on 1% gel

 

Gel:

Marker|RBS-GFP-Terminator Clone12

 

The test RD shows only one band indicating a successful cloning procedure. To be sure, the DNA will be used for another transformation hopefully leading to clean clones.

-          the plasmid DNA (RBS-GFP-TermC12) was analyzed on the 1%gel below.

2^nd round of RD of DarR and Ribiswitches A-D with new protocol

after Katrin Gunkas Überprotokoll

 

àFirst, the RDs of yesterday were analyzed on a 1% gel for complete digestion.

 

Gel:

Marker|DarR|Riboswitch 40/41|40/43|42/43|42/41|-|RBS-GFP-TermC12|-|part7after2ndRound2ndElution

 

The Riboswitch constructs shoe strong signals, DarR seems to be very sparsely left over.

 

àThe RDs were purified using the PCR purification kit (500µl PB and elution in 25µl pre-warmed water)

àThe purified DNA was analyzed with nanodrop

 

nanodrop:

	Concentration (ng/µl)	A260nm/A280nm	A260nm/A230nm
DarR biobrick	3.4	1.13	0.56
Riboswitch 40/41	9.1	1.63	1.08
Riboswitch 40/43	8.4	1.81	1.17
Riboswitch 42/41	5.7	2.26	1.00
Riboswitch 42/43	4.0	2.16	0.92

 

àOf the purified DNA, all 24µl left were used for the 2^nd round of RD with EcoRI FD

The 5 Reactions were pipetted like this

 

24µl DNA

16µl Mastermix:

4µl FD buffer

4µl EcoRI FD

8µl H2O

 

àIncubation at 37°C for 45 minutes

àFrozen away at -20°C

Miniprep of frozen plasmids from part7 and part8 in order to have DNA for cloning

NanoDrop:

Sample	ng/µl	A260nm/A280nm	A260nm/A230nm
Part7 mini	120.5	1.83	1.99
part8 mini	113.9	1.86	2.11

 

Incubation of Colony PCR master plates

è Plates taken out and put ot 4 °C (luckily not overgrown)

Enyzme test: ThermoScientific vs. NEB (Biobrick Assembly Kit)

è Since PstI digest was incomplete, it might be that the enzymes don’t work properly

è Test all enzymes in their preferred buffers

Test plasmid: plasmid part 1 purified on 1.7.13 à Linearization in single digest with EcoRI(-HF), XbaI, SpeI or PstI

-          Reaction according to test restriction digest to analyze clones after transformation

Component	Volume
Plasmid part 1 (2 ng/µl)	1 µl
Enzyme	1 µl
10x buffer	1 µl
dH2O	7 µl
Total Volume	10 µl

è Preparation of Mastermix:

Component	Volume
Plasmid part 1	15 µl
dH2O	105 µl

è Preparation of enzyme and buffer in Epi tubes

Company	Enzyme	Buffer	Activity
ThermoScientific	EcoRI	EcoRI buffer	100 %; recommended buffer
	EcoRI	Buffer 0	100 %
	XbaI	Tango	100 %
	SpeI	Tango	100 %
	PstI	Buffer 0	100 %
NEB (Biobrick Assembly Kit)	EcoRI-HF	CutSmart	100 %; recommended buffer
	EcoRI-HF	NEB2.1	100 %
	XbaI	CutSmart	100 %
	XbaI	NEB2.1	100 %; recommended buffer
	SpeI	CutSmart	100 %
	SpeI	NEB2.1	100 %; recommended buffer
	PstI	NEB3.1	100 %; recommended buffer
	PstI	NEB2.1	75 %

è then addition of 8 µl master mix

-          incubation at 37 °C for 2 h

-          gel run:

o   1 % agarose 1xTAE gel

o   3 µl 2 log ladder as a marker

o   Addition of 2 µl 5x LD to each reaction, then loading of 8 µl

o   Run at 100 V

o   EtBr staining, destaining in water

o   UV detection

 

è All digestions are partial; reaction of PstI in NEB2.1 is less complete than other digests

è Does not fit with complete digest of part 7 with EcoRI-HF in CutSmart buffer from 10.7.13; in this digest and in enzyme test different heating blocks were used à could they be the problem?

Yesterday´s Colony PCR of all constructs so far on 1% gel:

 

Gel:

Marker|Part8-plasmidDNA|GFP-Term-plasmid-DNA|Part8-w/o-insert-control|RBS-GFP-Term-C6|-7|-8|-9|-10|-11|-12|-13|-14|-15|-16|M

Marker|Part8-plasmidDNA|GFP-Term-plasmid-DNA|Part8-w/o-insert-control|RBS-GFP-Term-C17|-18|-19|part7-plasmidDNA|part7-w/o-insert control-C1|-2|-3|DarR-biobrick-C1|-2|RiboswitchC-biobrick|RiboswitchD-biobrick|M

è Clone 12 from RBS-GFP-Terminator transformation could be positive, at least partially

è Inoculation of clone 12 from RBS-GFP-Terminator transformation over night + MiniPrep

è Re-transformation of plasmid, since clone seems to bee impure (band for RBS-GFP-Terminator and RBS alone…)

Clones from RiboC and RiboD and DarR transformation are negative

 

Further RD of yesterday´s new protocol with part 7

Yesterday´s samples were analyzed with the nanodrop:

NanoDrop measurements from R.D. from yesterday:

Sample	ng/µl	A260nm/A280nm	A260nm/A230nm
purified part7 R.D. PstI	41.4	1.74	2.07
purified part7 2nd elute PstI	8.0	1.49	1.27
purified part7 R.D. EcoRI	4.2	1.35	0.77
purified part7 2nd elute EcoRI	2.0	0.84	0.51

Only the first elution of the RD with PstI looks promising. This sample was used for the further, second digest. For time reasons, the EcoRI digest was started in parallel to the 1% gel, that is supposed to confirm the complete digestion of part 7 by PstI. The following picture shows the progress of the digest at 30 additional minutes of RD (see yesterday).

Gel:

Marker|PstI RD Part 7

The digestion has not been completed, an additional step has to be added in the end.

2^nd round of digest:

àpipetted one reaction (40µl) after Katrin Gunkas Über-Protokoll (see yesterday) using EcoRI FD by Thermo Scientific and the FD buffer:

20µl DNA (all that was left)

4µl FD Buffer

4µl EcoRI FD

40µl reaction volume

àincubation at 37°C for 30 minutes

àsample put on ice and 3µl loaded on 1% gel.

àgel melted, samples lost

àconsidering the previous experience, the digest was continued with EcoRI. Additionally, to proceed the unfinished digest with PstI, that digest was added to the reaction.

The following things were added to the reaction:

2µl FD Buffer

3µl EcoRI FD

3µl PstI FD

20µl leading to a 60 µl reaction (minus 3µl on the gel)

àincubation at 37°C for 45 minutes

à3µl loaded on 1% gel (forgot to, loaded 6µl with PB of purification kitàdidn’t run)

Gel:

Needs to be redone, I suggest with the second elution, so we don’t lose too much DNA.

àpurification of the sample using the PCR purification kit, 500µl (different from protocol) PB buffer, 2x elution with 25µl pre-warmed HPLC water

nanodrop

Sample	ng/µl	A260nm/A280nm	A260nm/A230nm
purified part7 R.D. after 2nd round	27	1.71	1.59
2nd elution	3.4	2.71	1.06

àstorage at -20°C, “white sticker Ute-Box”

Inoculation of 4ml cultures for miniprep tomorrow

Three cultures of part 7 cryo-stocks from 11.6. and one culture of the RBS-GFP-Terminator construct Clone 12, which seems to be carrying at least partly the right construct (see today´s gel). Incubated in the Brutraum

New 1^st round Single digestion of DarR and Ribiswitches A-D with new protocol

after Katrin Gunka Überprotokol

9 µl PCR product

4 µl 10X Buffer F.D.

4 µl PstI F.D.

23µl dH₂O

incubated for 30 min at 37°C

Gel:

Wells as follows: DarR, Riboswitches 40/41, 40/43, 42/41, 42/43

rest of restriction stored in -20 freezer (for further digestion more enzyme has to be added)

..But where is DarR?...

Making 8 liters of expression culture of L. monocytogenes carrying gene for the DAC-domain for crystallization

-       50ml LB medium containing Ampicillin inoculated with cryo 1013 stock

-       incubation o/n at 37°C with agitation

-       measure OD₆₀₀

-       inoculation of 1l LB medium containing Ampicillin to OD₆₀₀=0,1

-       incubation for 2 hours at 37°C to an OD₆₀₀=0,5-0,9

-       induction of expression by adding 1ml of 1M IPTG

-       incubation for 12-14h at 16°C with agitation

-       measure OD₆₀₀

-       divide 1l of culture in twice 500ml

-       centrifugation at 5000 rpm for 10min at 4°C

-       resuspend the pellets in ice cold 20ml buffer W and take it in a falcon tube

-       pool both separated treated samples together

-       centrifugation at 5000 rpm for 15min at 4°C

-       discard supernatant

-       store pellet at -20°C

 

Protein purification

-       resuspend cells from one falcon (pellet from 40ml of expression culture) in 10ml buffer W

-       apply solution from each falcon twice to french press

-       centrifugation for 15min at 8500rpm at 4°C

-       transfer supernatant into ultracentrifugation tubes

-       Ultracentrifugation for 1h at 35000rpm at 4°C

-       Preparation of columns: add 1ml of 50% Strep-Tactin and equilibrate with 10ml buffer W

-       Divide supernatant received from ultracentrifugation into 2 equal volumes and fill it up to 30ml with buffer W

-       Load protein solution on prepared columns

-       Wash 5 times with each 2,5ml buffer W

-       Elute with 0,5ml (for eluate 1) or 1ml (eluate 2-4) of buffer E

 

Determination of protein concentration via Bradford test

-       Add 1ml of Bradford solution (1:5 dilution = 200ml Bradford + 800ml H2O) to 3µl of protein solution

-       Incubation for 5min at RT

-       Measure extinction at 595nm

-       Calculation: c= OD595 / Vx0,0536

 

Eluate	Absorption (nm)	C [µg/ml]
1 E1	0,078	0,49
2 E1	0,239	1,49
3 E1	0,092	0,57
4 E1	0,042	0,26
5 E1	0,075	0,47
6 E1	0,049	0,3
7 E1	0,053	0,33
8 E1	0,128	0,70
Total: 4,197 mg in 4ml
1 E2	0,143	2,57
2 E2	0,114	0,71
3 E2	0,169	1,05
4 E2	0,127	0,79
5 E2	0,061	0,38
6 E2	0,188	1,17
7 E2	0,121	0,75
8 E2	0,141	0,88
Total: 8,3 mg in 8ml

 

Dialysis of protein samples

-       Pool all samples from E1 (4ml)

-       Pool all samples from E2 (8ml)

-       Pool all samples from E3 (8ml)

-       Transfer eluates into dialysis bags

-       Put dialysis bags into 4500ml H2O + 500ml 10x dialysis buffer

-       Dialysis o/n with agitation

 

Preparation of plates from 500 ml LB^Cm plates (35 µg/ml Cm)

Further incubation of RBS-GFP-Terminator back-up plate

è Taken out in the evening (clones appear not green)

Cryostock of GFP-Term C1 overnight culture

stored at -80

Mini-prep of

Part7 C1

GFP-Term C1

RBS-GFP-Term C1

RBS-GFP-Term C2

RBS-GFP-Term C3

RBS-GFP-Term C4

RBS-GFP-Term C5

eluted in 30 µl HPLC

Nano Drop:

Sample	ng/ µl	260/280	260/230
GFP-Term gel ex	6.8	1.84	0.10
pSB1C3	15.6	1.72	0.15
part 7 C1	115.3	1.91	2.22
GFP-Term C1 mini	111.0	1.90	2.19
RBS-GFP-Term C1	285.7	1.89	2.26
RBS-GFP-Term C2	139.3	1.92	2.18
RBS-GFP-Term C3	157.6	1.88	2.04
RBS-GFP-Term C4	163.8	1.90	2.19
RBS-GFP-Term C5	155.0	1.91	2.20

DarRoperator PCR: 4 % gel run to check if PCR worked

 

Pouring of 4 % agarose-1xTAE-gel

1. Set waterbath to 70 °C and place gel chamber directly next to it.

2. Take 500 ml bottle (blue lid), add 4 g agarose and fill up to 100 ml with 1xTAE

3. Heat carefully in microwave (check every 5 – 10 sec if agarose does not … äh… überkochen… and mix by shaking bottle slightly (schwenken…) à Dissolving takes just a 2 – 4 min!

4. As soon as agarose is dissolved (clear solution, but many small bubbles from boiling and shaking making it turbid), place it in the waterbath (lid closed), wait for some time until bubbles are gone

5. Check again if agarose is still dissolved, if not, go back to step 3…

6. Pour gel rapidly.

 

Loading of

-          4 µl PCR reaction + 1 µl 5xLD

-          4 µl primer iGEM_36 + 1 µl 5xLD

-          4 µl primer iGEM_37 + 1 µl 5xLD

-          3 µl 2 log ladder (marker)

 

Run at 80 V

EtBr staining; destaining in water

UV detection

 

4% Gel with DarR operator:

Loading:

Lane 1: 2 log ladder; Lane 2: iGEM_36; Lane 3: iGEM_37; Lane 4: Negative control of PCR; Lane 4: PCR reaction tube 1; Lane 5: PCR reaction tube 2; Lane 6: PCR reaction tube 3; Lane 7: 2 log ladder

 

 

è PCR worked

è All reactions were pooled into one reaction

è Determination of concentration and purity (does actually not make sense…)

 

Sample	Concentration (ng/µl)	A260nm/A280nm	A260nm/A230nm
DarRop PCR product	1063.5	1.65	1.11

è Far too much since PCR reaction, that was not purified: Calculate with 100 ng!

 

Restriction Digestion of DarRop PCR product for ligation with pSB1C3 obtained from R.D+gel ex of part 7

-          More or less according to BioBrick Assembly Kit

 

Component	Reaction 1 µl PCR product	Reaction 5 µl PCR product	Reaction 10 µl PCR product
10x NEB2.1 buffer	5 µl	5 µl	5 µl
Eco-RI HF	1 µl	1 µl	1 µl
PstI	1 µl	1 µl	1 µl
PCR product	1 µl	5 µl	10 µl
dH2O	42 µl	38 µl	33 µl
Total	50 µl	50 µl	50 µl

è Reactions with 5 or 10 µl were incubated for 1 h at 37 °C

è Reaction with 1 µl was incubated for >1h at 37 °C

è Heat kill for all reactions: 20 min, 80 °C

 

-          Gel run

-          4 % agarose-1xTAE gel

-          Loading of

     4 µl pf PCR product + 1 µl 5x LD as a control

     8 µl of RD reactions + 2 µl 5x LD à just 8 µl of final sample (10 µl) loaded

     3 µl 2 log ladder as a marker

-          Run at 80 V

-          EtBr staining, destaining in water

-          UV detection

 

Gel:

Loading:

Lane 1: 2 log ladder; Lane 2: DarRop PCR product uncut; Lane 3: DarRop PCR product R.D. 1 µl; Lane 4: DarRop PCR product R.D. 5 µl; Lane 5: DarRop PCR product R.D. 10 µl

è PCR product still detectable for 5 and 10 µl reaction, but not for 1 µl reaction

è For 1 µl reaction probably band at bottom of lane à degradation?

è Use 5 and 10 µl reactions for cloning

è Reactions stored at -20 °C in pink box

 

Pooling of samples from gel ex on 4.7.13

- pSB1C3 vector from part 7

- GFP-Terminator insert

à determination of new concentration with NanoDrop

 

Test gel for plasmids and inserts

- plasmids for part 7 and GFP-Terminator C1 isolated in today’s MiniPrep

- plasmids part 1, 2, 3, 4 (purified on 2.7.13)

- GFP-Terminator insert after gel ex

- pSB1C3 vector obtained from RD and gel ex of part 7

- plasmid GFP-Terminator clone 1 (purified on 2.7.13)

-          1%-agarose-1xTAE gel

-          Loading of around 30 ng (or more) of plasmid DNA (0.5 µl for all samples; exceptions: pSB1C3 vector from part 7 à 2 µl, GFP-Terminator insert à 4 µl, filled up with dH₂O to 4 µl (exception: GFP-Terminator insert), then addition of 1 µl 5xLD

-          Marker: 3 µl 2 log ladder

-          Run at 100 V

-          EtBr staining, destaining in water

-          UV detection

 

Gel:

Loading:

Lane 1: 2 log ladder; Lane 2: part 1 uncut, Lane 3: part 2 uncut; Lane 4: part 3 uncut; Lane 5: part 4 uncut; Lane 6: part 7 (from today’s prep); Lane 7: gel ex pSB1C3 from part 7; Lane 8: GFP-Terminator C1 plasmid (former prep); Lane 9: GFP-Terminator C1 plasmid (today’s prep); Lane 10: gel ex GFP-Terminator construct from RD of GFP-Terminator C1 plasmid; Lane 11: 2 log ladder

 

è Part 7 is apparently impure à residual undigested plasmid though gel extraction was done (band of uncut plasmid has same height as pSB1C3 vector…)

 

Test Restriction Digestion of RBS-GFP-Terminator clones 1 – 5

-          Fermentas/ThermoScientific enzymes were used

For 1 reaction:

 

Component	Amount
Plasmid DNA	200 – 300 ng (1 – 1.5 µl in this case)
PstI	0.5 µl
EcoRI	0.5 µl
dH2O	Depends on plasmid amount (7 or 6.5 µl)
10x buffer O	1 µl
Total	10 µl

 

è Preparation of plasmid amount in 3 µl total volume:

Plasmid from…	…C1	…C2	…C3	…C4	…C5
Plasmid	1 µl	1.5 µl	1.5 µl	1.5 µl	1.5 µl
dH2O	2 µl	1.5 µl	1.5 µl	1.5 µl	1.5 µl

è Preparation of MasterMix

Component	Amount
PstI	3 µl
EcoRI	3 µl
dH2O	30 µl
10x buffer O	6 µl
Total	42 µl

 

è Add 7 µl to each reaction

-          Incubation at 37 °C for 1.5 h (no heat kill necessary in this case, since it’s just run on the gel and then thrown away)

-          Addition of 2.5 µl 5xLD

-          Agarose gel electrophoresis

1%-agarose-1xTAE gel

Loading of 10 µl of C1 RD (well run over…), thus loading of 8 µl of other RD’s

Loading of mixture consisting of 0.5 µl of all uncut plasmids + 3.5 µl dH₂O + 1 µl 5xLD as controls

Loading of 3 µl 2 log ladder as a marker

-          Run at 100 V

-          EtBr staining, destaining in water

-          UV detection

 

Gel for Test restriction digestion of RBS-GFP-Terminator clones 1 - 5

Loading:

Lane 1: 2 log ladder; Lane 2: C1 uncut; Lane 3: C1 R.D.; Lane 4: C2 uncut; Lane 5: C2 R.D.; Lane 6: C3 uncut; Lane 7: C3 R.D.; Lane 8: C4 uncut; Lane 9: C4 R.D.; Lane 10: C5 uncut; Lane 11: C5 R.D.; Lane 12: 2 log ladder

è Something strange of 100 bp got cloned…

Back-Up-Plates

è For all inoculated RBS-GFP-Terminator clones

è Incubation over day at 37 °C

MiniPlasmidPrep

è Clones 1 – 5 of RBS-GFP-Terminator construct

è Used buffer A4 without Ethanol added… concentrations after elution were quite low…

è Elution with 30 µl HPLC water (pre-warmed)

NanoDrop:

Sample	Concentration (ng/µl)	A260nm/A280nm	A260nm/A230nm
Clone 1	4.3	1.92	0.49
Clone 2	3.1	1.53	0.31
Clone 3	1.7	2.74	0.48
Clone 4	3.8	2.09	0.34
Clone 5	4.1	2.31	0.44

è Repeat MiniPrep…

PCR for DarRoperator

è As on 19.6.13

Component	1x Reaction	5x Master Mix
5x buffer HF	10 µl	50 µl
PhuS	1 µl	-
Primer iGEM_36	4 µl	20 µl
Primer iGEM_37	4 µl	20 µl
dNTPs	2 µl	145 µl
dH2O	29 µl	10 µl
Total	50 µl	245 µl à add 49 µl to each reaction

Control: 1µl dH₂O instead of PhuS polymerase

Protocol:

	Temperature	Time	No. of cycles
Initial Denaturation	98.5 °C	5min	1
Denaturation	98.5 °C	30s	10 cycles
Annealing	61.0°C	30s
Elongation	72.0°C	2min
Final Elongation	72.0°C	10min	1
Hold	15 °C	∞	1

è Reactions stored at -20°C in 50 ml Falcon tube

Ligation of Riboswitch inserts A, B, C, D with pSB1C3 backbone obtained from gel ex of dig. Part 7 plasmid

 

Ligation more or less according to BioBrick-Assembly-Kit

Ca. 20 ng of pSB1C3 vector ligated with ca. 20 ng of the different inserts

 

Reactions:

Component	RiboA (iGEM_42/43)	RiboB (iGEM_41/42)	RiboC (iGEM_40/43)	RiboD (iGEM_40/41)	DarR	w/o insert control	MasterMix
pSB1C3 vector (tube 3; 10.2 ng/µl)	2 µl	2 µl	2 µl	2 µl	2 µl	2 µl	14 µl
insert	3.5 µl (5.9 ng/µl)	1.5 µl (15.1 ng/µl)	2 µl (11.7 ng/µl)	3 µl (7.1 ng/µl)	3 µl (6.8 ng/µl)	-	-
dH2O/HPLC-H2O	11.5 µl	13.5	13 µl	12 µl	12 µl	15 µl	70 µl
Ligase	1 µl	1 µl	1 µl	1 µl	1 µl	1 µl	7 µl
10x ligation buffer	2 µl	2 µl	2 µl	2 µl	2 µl	2 µl	14 µl
Total	20 µl	20 µl	20 µl	20 µl	20 µl	20 µl	105 µl

è Prepare 5 µl of insert-HPLC water mix and add 15 µl of Master Mix

è Incubation for 20 min at RT

è Heat kill for 20 min at 80 °C

Transformation of E.coli DH5α competent cells

-          According to methods folder

             Transformation of

              part7-vector+Ribo A

              part7-vector+Ribo B

              part7-vector+Ribo C

              part7-vector+Ribo D

              part7-vector+DarR

              part7-vector w/o insert

              Control

-          Whole ligation mix used

-          Negative control: 20 µl dH₂O

-          Selection on LB^Cm plates (35 µg/ml Cm)

Inoculation of

RBS-GFP-Term C1, C2, C3, C4, C5

part7 C1

GFP-Terminator C1