Team:Tuebingen/Experiments

> 3A-Assembly

1μl 10x ligase buffer
0,5μl ligase
1μl ATP
0,51μl linearised vector
33,5μl insert 1
33,5μl insert 2

Method:

Mix all components
Incubate overnight at 4°C
Gelpurify using agarose gels

> Chemically Competent E. coli Cells

autoclave 250mL LB in Erlenmeyer beaker
plate cells from cell stock on agarose plate with appropriate antibiotics
inoculate one clone in 5mL of LB with antibiotics, grow at 37°C o/n
expand to 500mL and grow until OD600nm = 0.35
transfer into 50mL Falcon tubes
refrigerate centrifuge at 4°C, chill 2x 25mL 0.1M CaCl2 (Falcon tubes) and 5mL 0.1M CaCl2 with 10% Glycerol
spin down cells at 2000g for 10Min at 4°C
resuspend 5 Falcon cell pellets in 25mL ice cold 0.1M CaCl2
spin down at 2000g for 10Min at 4°C
resuspend pellet in 2x5mL (=10mL) ice cold 0.1M CaCl2 with 10% Glycerol
freeze 50-100μl aliquots
store at 20°C

> ColonyPCR

0,1μl forward primer (10μM)
0,1μl reverse primer (10μM)
5μl 2x Q5 Mastermix
4,8μl H2O

Method:

Pick a colony from plate and streak in a PCR tube. Mix components and add to tube.
Run PCR:
95°C 5:00min
95°C 0:45min
53°C 0:45min
72°C 1:30min
72°C 10:00min
4°C inf

> DoubleDigest Restriction of gBlocks and BioBricks

2μl 10x buffer (corresponding to enzymes)
0,5μl restriction enzyme 1
0,5μl restriction enzyme 2
1μg DNA
fill up to 20μl with H2O

Method:

mix all components
incubate at 37°C for 2h
heatinactivate enzymes at 80°C for 10min or use for gel purification

> Gelpurification

This is a modified protocol for the Wizard SV Gel and PCR CleanUp System Kit from Promega

Following electrophoresis, excise DNA band from gel and place gel slice in a 1,5ml microcentrifuge tube
Add 10μl Membrane Binding Solution per 10mg of gel slice. Vortex and incubate at 5060°C until gel slice is completely dissolved
Insert SV Minicolumn into Collection Tube
Transfer dissolved gel mixture to the Minicolumn assembly. Incubate at room temp. for 1 minute
Centrifuge at 16,000xg for 1 min. Discard flowthrough and reinsert Minicolumn into Collection tube
Add 700μl Membrane Wash Solution (ethanol added). Centrifuge at 16,000xg for 1 min
Discard flowthrough and reinsert Minicolumn into Collection tube
Repeat this step with 500μl Membrane Wash Solution. Centrifuge at 16,000xg for 5 min
Empty the Collection Tube and centrifuge the column assembly for 1,5 min
Leave the tubes open for 10 min (to let any rest of ethanol evaporate)
Carefully transfer the Minicolumn to a clean 1.5ml microcentrifuge tube
Add 30μl of NucleaseFreeWater (65°C) to the Minicolumn. Incubate at room temperature for 10 min
Centrifuge at 16,000xg for 1 min.
Incubate sample at 65°C for 5min
Discard Minicolumn and store DNA at 4°C or 20°C

> Ligation

1μl 10x ligase buffer
0,5μl ligase
1μl ATP
0,51μl linearised vector
7μl insert

Method:

Mix all components
Incubate overnight at 4°C
Gelpurify using agarose gels

> MutagenesisPCR

200ng template
1μl forward primer (2mM)
1μl reverse primer (2mM)
1μl Phu polymerase
1μl dNTPs
10μl 5x Buffer
to 50μl with H2O
Mix components
Run thermocycler:
98°C 0:30min
98°C 0:45min
55°C 0:45min
72°C 7:00 min
72°C 1:00 min
4°C inf

> Oligo Annealing

2μl oligo 1
2μl oligo 2
96μl elution buffer

Method:

Mix all components
incubate for 10 minutes at 95°C
take tube with heat block out of heat and let it cool down to room temperature

> Smallscale plasmid preparation

Preparation was done using the PureYield™ Plasmid Miniprep System by Promega´

Pick colonies for overnight cultures in 23ml LB medium with antibiotic grow overnight
Spin down 1,5ml bacteria at maximum speed for 30 minutes, discard supernatant and add further 1,5ml, spin again and discard supernatant
Resuspend the bacteria pellet in 600μl H2O
Add 100 μl Cell Lysis Buffer, invert six times
Add 350 μl cold Neutralization Solution, mix thoroughly by inverting
Centrifuge 30s at maximum speed
Transfer supernatant to PureYield Minicolumn in collection tube
Centrifuge for 30 sec, discard the flow-through
Add 200 μl Endotoxin Removal Wash(ERB), centrifuge for 30sec, discard the flow-through.
Add 400 μl Column Wash Solution(CWC), centrifuge for 30sec, discard the flow-through.
Place column in eppendorf tube, add 30 μl Elution Buffer to minicolumn matrix, incubate for 1 min
Centrifuge column for 30s at maximum speed, flow-through contains plasmid DNA
Measure the DNA concentration using a Nanodrop

> Testrestriction

0,25μl Enzyme 1
0,25μl Enzyme 2
1μl 10x buffer (corresponding to enzymes)
200-300ng DNA
Fill up with water to 10μl.

Method:

Incubate at 37°C for 1h
Run on 1% agarose gel

> Transformation of Bacteria

50μl chemically competent E. coli
35μl overnight ligation mix OR 1ng purified plasmid DNA

Method:

Thaw bacteria pellet on ice
add ligation mixture or purified plasmid to bacteria
incubate for 20min on ice
heat shock bacteria for 45s at 42°C
incubate bacteria on ice for 2min
add 700μl LB medium without antibiotics
incubate for 1h at 37°C
spin down bacteria at 2g for 2min
discard supernatant by tipping the tube
resuspend bacterial pellet in leftover supernatant (50100μl)
streak bacteria onto LBagar plates with antibiotics and incubate overnight

> Transformation of Yeast

ca. 10ml YPD
100μl OneStep buffer
20μg ssDNA
100-500 ng plasmid DNA
fresh YPD selective plate

Method:

Scrape some yeast cells off a fresh YPD plate and inoculate in liquid YPD (= culture)
Thaw ssDNA (e.g. salmon sperm DNA) and heat for 10 min at 95 °C, then chill on ice
Pellet 1 mL of culture by centrifugation at > 13.000 rpm
Discard supernatant and resuspend cells in 100 μL OneStep buffer, vortex heavily
Add 20 μg ssDNA (10 μL of 2 mg/mL) and 100 500 ng plasmid DNA to be transformed
Vortex and incubate at 45 °C for 2 h
Add 1 mL YPD, mix and spin 10 sec at full speed, discard supernatant
Resuspend cell pellet in 1000 μL YPD and plate 100 μL directly on appropriate selective plates
Colonies appear after 2 days of incubation at 30 °C

> TBE

1M tris
0,85M B(OH)3
2mM EDTA
pH=8,0

> SDS-PAGE running buffer

20mM Tris/HCl pH 7,6
250mM Glycin
0,1% (w/v) SDS
in ddH2O

> 1x Laemmli

50mM Tris pH 6,8
1,25mM EDTA pH 8
12,5% (v/v) Glycin
2% (w/v) SDS
50mM DTT
2,5% (v/v) -Mercaptoethanol
0,025% (w/v) Bromphenolblau
in ddH2O

> LB

20g Lennox Broth
to 1l with H2O

> LB/Agar

35g LB-Agar (Lennox)
to 1l H2O

> SC-media

2g yeast nitrogen base w/o amino acids
0,25g synthetic complete drop-out mix
16,5mg adenine-sulfate
to 300ml H2O
adjust pH to 5.6
add agar if needed

> synthetic complete drop-out mix

1g adenine hemisulfate
1g arginine-HCl
1g histidine HCl*
1g isoleucine
2g leucine*
2g lysine-HCl
2g methionine*
1,5g phenylalanine
1g serine
1g threonine
1,5g tryptophane*
1g tyrosine
0,6g uracil*
4,5g valine
for dropout mix, omit appropriate components, labelled with *
combine ingredients and mix thoroughly

> YEP (yeast extract peptone)

3g yeast extract
6g peptone
30mg adenine hemisulphate
300ml H2O
if needed, add 5g agar

> 20% Glucose/Galactose/raffinose

20g of appropriate sugar
100ml H2O

> antibiotics concentration

chloramphenicol 34µg/ml
ampicillin 100µg/ml

> One-step buffer

0,2M LiAc
40% PEG4000
100mM DTT
sterile filtrated

SDS-Gels

> 12% separation gel

40,5% acrylamide (30%)
0,375M Tris (pH 8,8)
1% SDS (10%)
1% APS(10%)
0,1% TEMED

> 5% stacking gel

17% acrylamide (30%)
0,125M Tris (pH 6,8)
1% SDS (10%)
1% APS (10%)
0,1% TEMED

Ni-NTA buffers

All buffers are from the Qiagen "Ni-NTA Agarose Purification of 6xHis-tagged Proteins from E. coli under Native Conditions - Quick-Start"-protocol.

> Lysis buffer (pH = 8)

50 mM NaH2PO4
300 mM NaCl
10 mM Imidazole

> Washing buffer (pH = 8)

50 mM NaH2PO4
300 mM NaCl
20 mM Imidazole

> Elution buffer (pH = 8)

50 mM NaH2PO4
300 mM NaCl
250 mM Imidazole

Experiments

> Agarose Gels

cast gels
load samples with loading dye
run gels at 120 V

> Expression

pETue-NAGA-Intein was transformed into E. coli BL21(DE3)

grow culture to OD_600 = 0.3

induce with 0.2 mM IPTG

express O/N at room temperature

centrifuge at 6000 g for 30 minutes at 4°C

resuspend pellet in 10 ml lysis buffer

sonicate for a total of 3 minutes (40% amplitude, 1 s intervals)

> Fluorescence assay

Cells were measured undiluted or in 1:2 dilution in 96 well plates (black, flat bottom)

Following excitation and emission wavelengths were used for the different fluorescent proteins:

GFP

Exc: 488 nm

Em: 520 nm

Dronpa

Exc: 503 nm

Em: 535 nm

RFP

Exc: 584 nm

Em: 616 nm

Fluorescence was measured with Tecan plate reader (Infinite M200) and analysed with Tecan i-control v1.10

> Fluorescence Microscopy

Sample preparation

A thin layer of low melting agarose was supplied on glass slides

10 µl Yeast cells grown overnight were inoculated in the solidified agarose

samples were used for microscopy after at least 30min incubation at 30° in a humid environment

Microscopy Setup

We used a Zeiss LSM 710 microscope with a Plan-Apochromat 63x/1.40 Oil DIC M27 objective. All pictures were taken with one active fluorescent channel and brightfield channel. Dronpa was observed by using a 514 nm laser (2% power) for excitation, and RFP was observed by using a 633 nm laser (2% power) for excitation

Dronpa Illumination

Illumination of dronpa was performed in vivo using a 488 nm argon laser at 50% power for off switching and a 405 nm for on switching of fluorescence. The illumination of cells was either performed using continuous illumination for 10 to 20 seconds or by performing a line scan with the illumination laser.

> Luciferase Assay

Grow overnight culture of yeast: Induction or repression of luciferase expression by addition of different galactose/glucose/raffinose concentrations for pGal or pSUC promoters.

NanoLuc luciferase protocol: add 25 µl cell culture, 25 µl reconstituted luciferase assay reagent and 50 µl PBS in 96 well plate (white, clear bottom)

Firefly luciferase protocol: mix either cell suspension or cell lysate 1:1 with reconstituted luciferase assay reagent

Luciferase activity measured with Tecan plate reader (Infinite M200) and analysed with Tecan i-control v1.10.

> Fluorescent-peptide-coupling Assay

incubate 100 µl of Ni-NTA purified eluate with 10 µl (10 mg/ml, 7,90 mM) peptide solution for 24 or 48 hours at 5°C or room temperature

dilute with 110 µl of 2x Laemmli buffer

incubate at 95°C for 10 minutes

load onto SDS polyacrylamide gel

> MALDI-TOF mass spectrometry

Preparation and trypsin digest:
cut bands from SDS polyacrylamide gel

cover gel piece with Buffer2 for 30 min to discolor

cover with acetonitrile (ACN) for 10 min to dehydrate

cover with Buffer1 containing 10 mM DTT to reduce for 45 min

cover with Buffer1 containing 55 mM iodoacetamide (IAA) to alkylate for 30 min

cover with Buffer1 for 15 min to wash

cover with Buffer2 for 10 min to wash

cover with 100% ACN for 10 min to dehydrate

cover with 20 µl Buffer1 containing 100 ng Trypsin

digest for 5 h at 37 °C

stop reaction by addition of 2 µl of 10% TFA

extract peptides by incubation for 30 minutes
Buffer1: 50 mM ammoniumbicarbonat (AB) Buffer2: 70% 50 mM AB, 30% ACN Measurement:
mix 1 ml of sample with 1 µl of gentisic acid matrix solution

pipette unto target

wait for crystallisation

measure with MALDI-TOF

analyse data using FlexAnalysis und FlexControl
The result score is calculated as Score = -log10(P).

> Ni-NTA Purification

Pellet
centrifugation of the bacterial culture for 10 min at 8000 rpm at 4 °C

discard the supernatant
Lysis
per 1g wet weight of the pellet add 4ml lysis buffer

fill up to 0.5 mg/ml with lysozyme

incubation on ice for 2 h

centrifuge for 30 min at 10000 g at 4 °C
Column
equilibrate the column with 0.5 ml Ni-NTA (0.25 ml bed volume)

add 0.5 ml lysis buffer and slightly centrifuge the buffer through the column, it has to stay wet (do twice)

pump the cell lysate (supernatant) for 1 h at ice through the column
Wash
twice with 1.25ml wash buffer

four times with 0.5 ml elution buffer

recover the supernatant

Load a sample of every step in an SDS gel.

> Purification

centrifuge lysate at 15,000 g for 30 min

purify supernatant with Ni-NTA beads (native conditions, gravity flow)

elute once with 500 µl elution buffer

> SDS-page

Cast gels with composition as described (Link bitte einfügen zu der Zusammensetzung)

Load samples with SDS-PAGE loading dye

Run gels at 150V until front is run through the gel