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Revision as of 21:37, 4 October 2013

Cloning

Colony PCR

Colonies were picked with pipet tips and dipped into the PCR mixture.

Ingredient	amount [µl]
Overnight Culture	Top of a pipet tip
10x PCR buffer	2.0
MgCl2 (25mM)	2.0
Forward primer (10µM)	0.2
Reverse primer (10µM)	0.2
dNTPs (10mM)	1.0
Polymerase	0.5
Sterile water	ad 10.0
Total	10.0

The following PCR program was adjusted according to annealing conditions.

Cycles	temperature [°C]	Time [min:s]
1	98	2:00
35	98	0:05
	68 (annealing)	0:15
	72	1:00 per kbp
1	72	10:00
1	12	inf

Agarose gel electrophoresis

To separate DNA fragments of different sizes, agarose flat-bed gels were casted in various concentrations (0.6% to 2% agarose in 0.5x TAE buffer). By applying an electrical field (10 to 20 V/cm), these gels were used for analytical or preparative separation. Therefore, the desired amount of agarose was boiled up in 0.5x TAE buffer until it was completely dissolved. After the solution cooled down to 50 to 60°C, it was poured into a flat-bed tray and ethidium bromide (EtBr) solution (0.5 µg/ml final concentration) was added. Finally, a comb was inserted to create pockets while solidification. The gel was put into the running buffer (0.5x TAE buffer) before the DNA mixture with loading buffer was loaded into the pockets and separated by electrophoresis. As ethidium bromide intercalates with the DNA (GC nucleotides) the resulting DNA-EtBr-complexes are visible under UV irradiation at 265 nm. As standard ladder for fragment sizes, 1kb+ ruler, 2-log ladder or 50 bp ladder were used.

Gibson assembly

For Gibson assembly the following mixtures were prepared:

A) ISO buffer

amount	ingredient	remarks
1.5 g	PEG-8000
3 ml	Tris-HCl (1 M, pH 7.5)	dissolve 12.1 g Tris in 100 ml dH₂O, adjust pH to 7.5 with conc. HCl
300 µl	DTT (1 M)	dissolve 1.54 g DTT in 10 ml dH₂O
150 µl	MgCl₂ (2 M)	dissolve 4.06 g MgCl₂ in 10 ml dH₂O
300 µl	NADNa (100 mM)	dissolve 0.02 g NADNa in 300 µl dH₂O
4 x 60 µl	dNTPs (100 mM, each)
up to 6 ml	dH₂O

aliquot á 350 µl

B) Assembly master mix

Work on ice!

volume	ingredient
690 µl	dH₂O
320 µl	ISO buffer
160 µl	Taq ligase (NEB, 40 U/µl)
20 µl	Q5 Polymerase (NEB, 2 U/µl)
10 µl	T5 Exonuclease (NEB, 0.64 U/µl)*

dilute 3.2 µl T5 Exonuclease (10 U/µl) in 46.8 µl 1x T5-buffer

aliquot á 15 µl

C) Calculation for Gibson cloning

Excel template kindly provided by the iGEM Team Freiburg 2013
Gibson assembly was prepared according to calculation (see file above). The mixture was incubated at 50°C for 60 minutes and purified by isopropanol precipitation. The Gibson constructs were then transformed into competent bacteria.

Biobrick assembly

[http://ginkgobioworks.com/support/BioBrick_Assembly_Manual.pdf BioBrick_Assembly_Manual.pdf]

Restriction digests

For enzymatic digests, the following formula was used if bovine serum albumin (BSA) was not part of the buffer, which was the case for NEB buffer 3.0:

... µl DNA (~500ng)
5µl BSA
5µl buffer (for example NEBuffer 2)
2 x 1.5µl enzymes (for example EcoRI-HF & PstI)
... µl water

=> 50µl in total

The mixture was incubated at 37°C for 1h or up to 1:30h and analyzed by gel electrophoresis.
In case BSA was already part of the buffer, the following recipe was used:

... µl DNA (~500ng)
2µl buffer (e.g. NEB CutSmart buffer)
1µl restriction enzymes
water ad 20µl

The mixture was incubated at 37°C for 1h or up to 1:30h and analyzed by gel electrophoresis.

Extraction of DNA fragments from an agarose gel

After gel electrophoresis, the gel parts containing the desired DNA fragement size were excised from the agarose gel with a clean and sharp scalpel. For DNA extraction the QIAquick® Gel Extraction Kit was used. Following the instrucion manual all necessary solutions have been prepared. The slices were weighted and accordingly Buffer QG added to the gel (for 1 slice 600 μl; for 2 slices 900 μl). The tubes with the mixture were incubated at 55°C until the gel slices had completely dissolved. In between samples were vortexed, if fragement sizes were not too large. Since the color of the solution never changed, no pH adjustments had to be done. In the next step isopropanol was added (for 1 slice 200 μl; for 2 slices 300 μl) to the sample and mixed. For each sample a QIAquick spin column was placed into the vacuum manifold and the sample decanted to it to bind DNA. Vacuum was applied until the whole sample has been drawn through the column. For further purification samples were washed with 500 μl buffer QG and vacuum was applied. Afterwards 750 μl Buffer PE were added to the samples, let stand for 2-5 minutes and vacuum applied. To remove residual wash buffer, the columns were placed into collection tubes and centrifuged for 1 min at 17,900 x g (13,000 rpm). Finally, the columns were put into clean 1.5 ml microcentrifuge tubes and 20-30 μl water (55°C) were added to the center of the column. After letting the column stand for 1 minute it was centrifuged for 1 minute to collect the elute.

For larger DNA fragments, the QIAEX II® Gel Extraction Kit was used (suitable for 40 bp to 50 kbp). Solutions were prepared as advised in the instruction protocol. DNA bands were excised from the agarose gel with a clean and sharp scalpel and transfered to a microcentrifuge tube. After weighing the slices, buffer QX1 was added according to the DNA fragment size (6 volumes for <100 bp; 3 volumes for 100 bp – 4 kb; 3 volumes with 2 volumes of water for >4 kb). QIAEX II was vortexed for 30 seconds to resuspend the agent and added to the samples (10 μl QIAEXII for ≤2 μg DNA; 30 μl for 2–10 μg DNA; and an additional 30 μl for each additional 10 μg DNA). Subsequently the solution was mixed and incubated at 50°C for 10 min to solubilize the agarose and bind the DNA. Since we mainly used this kit for larger fragments, the resuspended pellet was only inverted and flicked to avoid shearing forces. We did not observe changes in color, that is why no pH adjustment were done. Afterwards, samples were centrifuged for 30 seconds and supernatant carefully removed with a pipet. The pellet was washed with 500 μl buffer QX1 to remove residual agarose contaminants and resuspended by inverting and flicking. Then samples were centrifuged for 30 seconds and all supernatant residues removed with a pipet. The pellets were washed twice with 500 μl buffer PE to remove salt contaminants and resuspended by inverting and flicking. Then samples were centrifuged for 30 seconds and supernatant removed with a pipet. Pellets were air-dried for 10-15 minutes until they became white. To elute the DNA, 20 μl of water were added and pellet resuspended by flicking. After incubation according to the DNA fragment size (5 minutes at room temperature (15–25°C) for ≤4 kb; 5 minutes at 50°C for 4–10 kb; 10 minutes at 50°C for >10 kb), samples were centrifuged for 30 seconds. Supernatant, which contained the purified DNA, was pipetted into a clean tube.

Ligation

For ligation of DNA fragments, the mixture was prepared as follows:

... µl DNA (=<200ng in total-> 20-70ng Backbone)

2 µl buffer

1 µl ligase (T4)

... µl water => 20 µl in total After an incubation for 2 minutes at room temperature, or for 1h or over night at 4°C, the product was loaded on an gel for electrophoretic separation of the DNA fragments.

Nucleotide Removal

For removal of excess nucleotides after enzymatic reactions, the QIAquick® Nucleotide Removal Kit (QIAGEN) was used. All solutions were prepared as recommended by the manufacturer. Ten volumes of Buffer PNI were added to 1 volume of the sample and mixed. The QIAquick spin column was placed in a provided 2 ml collection tube and sample decanted to it. The tube was centrifuged at 6000 rpm for 1 minute. Flow-through was discarded and the column placed back into the collection tube. Afterwards, 750 μl Buffer PE were added, incubated for 2-3 minutes and centrifuged for 1 min at 3800 x g (6000 rpm). Flow-through was discarded and the column placed back into the collection tube. The column was centrifuged for an additional 1 min at 17,900 x g (13,000 rpm) to remove residual buffer. Finally, the column was put into a clean 1.5 ml microcentrifuge tube and 20-30 μl water (55°C) was added to the center. After 1 minute incubation the sample was centrifuged for 1 minute at 17,900 x g (13,000 rpm). The 1.5 ml tube now contained the eluted and purified DNA.

Purification of PCR products

To purify PCR products the QIAquick® PCR Purification Kit (QIAGEN) was used. Therefore, all solutions were prepared as recommended by the manufacturer. Five volumes of buffer PB were added to 1 volume of the PCR reaction before mixing. Since no changes in color were observed the pH was not adjusted. A QIAquick column was put into a vacuum manifold and the sample was decanted to it for binding the DNA. Vacuum was applied until all liquid residues were drawn through the column. Afterwards 750 μl buffer PE were added to wash the column and vacuum applied. Te remove residual wash buffer the QIAquick column was put into the provided 2 ml collection tube and centrifuged for 1 min at 17,900 x g (13,000 rpm). Finally, the column was put into a clean 1.5 ml microcentrifuge tube and 20 ml water (55°C) were added to the center to elute the DNA. After 1 minute incubation the tube containing the column was centrifuged for 1 minute. The microcentrifuge tube now contained the eluted DNA.

For small amounts of DNA PCR products (cleanup of up to 5 μg PCR product (70 bp to 4 kb)) the MinElute® PCR Purification Kit (QIAGEN) was used. All steps were conducted and solutions prepared as specified in the protocol. Five volumes of buffer PB were added to 1 volume of the PCR reaction and mixed. Since no changes in color were observed the pH was not adjusted. A MinElute column was put into a vacuum manifold and the sample was decanted to it for binding the DNA. Vacuum was applied until all liquid residues were drawn through the column. Afterwards 750 μl buffer PE were added to wash the column and vacuum applied. Te remove residual wash buffer the QIAquick column was put into the provided 2 ml collection tube and centrifuged for 1 min at 17,900 x g (13,000 rpm). The MinElute column was placed into a clean 1.5 ml microcentrifuge tube and 20 ml water (55°C) were added toe the center to elute the DNA. After 1 minute incubation the tube containing the column was centrifuged for 1 minute. The microcentrifuge tube now contained the eluted DNA.

Plasmid-DNA isolation

Miniprep
Plasmids were isolated by the use of the QIAprep® Spin Miniprep Kit. According to the instructions included, all necessary solutions were prepared and steps conducted. First, overnight cultures (1-2 ml) have been pelleted by centrifugation at >8000 rpm (6800 x g) for 3 min at room temperature (15–25°C). Supernatant was discarded and pellet resuspended in 250 μl (procedure 2: 200 μl) buffer P1 in a microcentrifuge tube. Afterwards 250 μl (procedure 2: 400 μl) Buffer P2 were added and the tube inverted 4-6 times to mix the solution until it became clear. The sample was lysed for 2-3 minutes before adding 350 μl (procedure 2: 300 μl) buffer N3 and inverting the whole solution 4-6 times. Following the neutralization, the sample was centrifuged for 10 minutes at 3,000 rpm (~17,900 x g) in a table-top microcentrifuge. Supernatant was decanted to a QIAprep spin column and vacuum applied to the manifold until the solution was drawn through the column. To wash the column 500 μl Buffer PB were added and again vacuum applied. In addition the QIAprep spin column was washed by adding 750 μl Buffer PE and applying vacuum. To remove residual wash Buffer the column was transfered to a collection tube and centrifuged for 1 min. Finally, the column was put into a clean 1.5 ml microcentrifuge tube and 20-30 μl water (heated up to 55°C) added to the center of the column. After 1 minute the tube containing the column was centrifuged for 1 minute to collect eluted DNA.

Midiprep
For larger amounts of bacterial cultures the QIAGEN® Plasmid Plus Midi Kit was used to isolate plasmids. According to the instructions included, all necessary solutions were prepared and 25 ml bacterial culture harvested after 12-16 hours incubation. The sample was centrifuged at 6000 x g for 15 min at 4°C and pelleted cells were completely resuspended in 2 ml Buffer P1. Then, 2 ml buffer P2 were added and mixed thoroughly by inverting until the lysate appeared viscous. The solution was incubated at room temperature (15–25°C) for 3 min. The QIAfilter Cartridge was placed into a new tube. Afterwards, 2 ml buffer S3 were added to the lysate and mixed by inverting 4-6 times. The lysate was transfered to the cartridge and incubated at room temperature for 10 minutes. During incubation the QIAGEN Plasmid Plus spin column was put into the vacuum manifold and tube extenders inserted into each column. The plunger was inserted gently into the QIAfilter Cartridge and the lysate filtered into the tube. Thereafter, 2 ml buffer BB were added to the cleared lysate and mixed by inverting 4-6 times. Lysate was then transfered to a QIAGEN Plasmid Plus spin column on the vacuum manifold. Vacuum was applied until all liquid remains were drawn through the column. To wash the DNA 700 μl buffer ETR was added and vacuum applied. Residual wash buffer was removed by centrifugation of the column at 10,000 x g (9,700 rpm) for 1 min in a tabletop microcentrifuge. The column was put into a clean 1.5 ml tube. Finally, 200 μl water (55°C) was added to the center of the spin column. The column was let stand for 1 minute and centrifuged for 1 minute to collect the eluted DNA.

Preparation of large plasmids
For isolation of plasmids larger than 15 kb in size, 7 ml bacterial culture were harvested after 12-16 hours of growth in appropriate medium. Samples were centrifuged at 6000 xg for 20 min at 4°C. Supernatant was discarded and cell pellet resuspended in 200 µl of Buffer P1 (QIAGEN). Afterwards, 400 µl of Buffer P2 were added, mixed thoroughly by inverting until the lysate appeared viscous. Solution was incubated at room temperature (15-25°C) for 2 min. Afterwars 300 µl of Buffer P3 were added to stop lysis. Cell debris and Proteins were pelleted by centtrifugation at ~ 17,900 x g for 30 min at 4°C. Supernatant was carefully transferred into a fresh 2ml eppendorf centrifuge tube and isopropanol purification of DNA was conducted.

Sequencing

Samples were sent to [http://www.gatc-biotech.com/en/index.html GATC Biotech] for sequencing.

Preparation of competent E. coli Top10 and DH10beta

Electrocompetent cells
Day 1
Autoclaving of following materials:

4x100 ml and 1x50 flasks containing 50 mL LB

400 mL Millipore Water (stored in the fridge after autoclaving)

100 mL 10% glycerol (stored in the fridge after autoclaving)
The 50 mL flask was then inoculated with cells and grown overnight at 37°C and 240 RPM. Day 2
On the second day 5 mL of the overnight culture were added to each of the four flasks containing 100 ml LB and incubated at 37°C with vigorous shaking until an OD 600nm between 0.5 and 1.0. was reached. Afterwards the log-phase culutures were transfered to tubes and placed on ice. The centrifuge was cooled down to 4°C and filled tubes inserted. After centrifugation for 15 mins at 2000g (3500 RPM) at 4°C the supernatant was discarded and pellets resuspended with 30 ml ice-cold sterile water. This washing step was then repeated again. Afterwards tubes were centrifuged for 15 mins at 2000g (3500 RPM) at 4°C, supernatant removed and pellet resuspended in 10ml cold 10% glycerol. The solution was transfered to 15 ml centrifuge tubes and stored on ice for 15 minutes. Following, the tubes were centrifuged for 15 mins at 2000g (3500 RPM) at 4°C. Supernatant was removed and 500 μl of 10% glycerol added. This stock solution was used for preparation of 100μl aliquots (microcentrifuge tubes on ice). Cell suspensions were shock frozen with liquid nitrogen and stored at -80°C. Electroporation: In preparation for the electroporation 7mm x 100mm round-bottom culture tubes were used at room temperature. SOC recovery medium was placed in a 37°C water bathe and selective plates pre-warmed at 37°C for 1 hour. Meanwhile the electroporation cuvettes (1 mm) and microcentrifuge tubes were put on ice. Electrocompetent were thawed on ice and mixed by flicking and inverting. Afterwards, XXX μl of the cells were transferred to an ice chilled microcentrifuge tube and Plasmid DNA solution added (according to concentration measured at nanodrop). The mixture was transferred to a chilled cuvette. The electroporation was conducted using the following conditions: BTX ECM 630 and Bio-Rad GenePulser electroporators: 2.1 kV, 100 Ω, and 25 μF. With a time constant of ~2.6 milliseconds. 975 µl of 37°C SOC medium were added immediatly to the cuvette, mixed and transferred to the round-bottom culture tubes. After shaking at 250 rpm and 37°C for 1 hour, cells were diluted and plated on a pre-warmed selective plate. Finally, the plates were incubated overnight at 37°C.

Protein detection

Thin Layer Chromatography

TLC was carried out on silica-gel as immobile phase and Dichloromethane as mobile phase. For the procedure a 50 ml beaker was filled with ~15 ml Dichloromethane and let stand for about 10 minutes (in order to let the Dichloromethane-vapor fill the beaker). The TLC plate, coated with silica-gel was spotted with sample 0.5 to 1 cm above the lower edge and placed in the beaker. TLC was run until the solvent front was at two thirds of the TLC plate. As Indigoidine is light-sensitive, the beaker was covered with aluminium foil in order to prevent direct light irradiation.

Purification of Indigoidine and tagged constructs

1 ml of IPTG-induced, blue culture was spun down at full speed (14,000 rpm) for 20 minutes, washed in 1 ml of methanol and centrifuged once more for 5 minutes at 14,000 rpm. Methanol was discarded and samples were dissolved in 200-400 µl DMSO.

Purification of Delftibactin

Centrifugation of cultures at 3750 rpm for 30 min
Retrieve supernatant
Add 20 g/L HP-20
Stirr at RT for 2 h
Filtration with Buchner funnel to retain HP-20
Wash with 400 ml ddH₂O
Elution with 400 ml Methanol
Evaporated methanol with rotary evaporator
Resuspended in 2 ml 50 methanol : 50 ddH₂O

Mass spectrometry

Cell preparation

For analysis by mass spectrometry, 3 ml of LB media containing chloramphenicol (1 micromolar) was inoculated with transformed bacterial cells (expression of the NRPS tested before by SDS-PAGE). Cultures were incubated at 37°C overnight until OD₆₀₀ ~ 0.6. After centrifugation for 30 minutes at 3750 rpm and 4°C, supernatant was discarded and pellet washed with M9 minimal media. Again samples were centrifuged and resuspended in 50 ml M9 minimal media. Following an incubation time of 12 hours (OD600=0.6) cells were induced with IPTG (1 mM).

To assess product levels over time, 7.5 ml samples were taken at several time points (8.5 hrs, 12 hrs, 24 hrs and 48 hrs). Each sample was spun down for 45 minutes at 3750 rpm and 4°C, and supernatant was separated from pellet.

Pellet was resolubilzed in 500 µl 1x PBS and lysed by ultra-sonification (3x20 seconds; on ice). Finally the sample has been centrifuged and supernatant decanted to 1.5 ml tubes. Frozen with nitrogen the sample was handed over for mass spectrometry.

Supernatant was frozen with nitrogen and lyophilized for 12 hours ( at ~-45°C and ~0,1 mbar) to remove liquid residues. The lyophilisate was resuspended in 500 microliters methanol and vortexed. Supernatant was transferred to a 1.5 ml tube, which was frozen in liquid nitrogen. The sample was then handed over to mass spectrometry for further analysis.

Data acquisition

Sample taking

Two empty 3.2 mm die-cut sheets were prepared from Whatman 903 filter paper. Afterwards, 5 µl of the sample were pipetted on the sheets and dried in the fridge overnight.

Sample preparation
The dried sheets were extracted for ca. 20 min with 100 μl solution 1. Then the polypropylen microtiter plates were centrifuged for ca. 2 minutes at 4000 rpm. Supernatant was vaporized at 55°C for 30 minutes at the blowing station until all liquid residues were removed. Subsequently the plate was warmed up for 1 minute at a cabinet dryer (65°C). After drying 60 μl of solution 2 were added and the mircotiter plate tightly closed with micromates. The plate was shaken gently and heated up again for ca. 15 minutes at the cabinet dryer (65°C). Micromates were drawn off and supernatant vaporated on heating plates (55°C surface temperature) with a constant air stream. Again no liquid residues should be remaining. Samples were heated up for 1 minute in the cabinet dryer (65°C). Finally 100 μl solution 3 were added (it is possible to dilute the sample up to factor 3 with solution 3). To avoid evaporation losses, the microtiter plate was covered with aluminium foil.

SDS-PAGE

1 ml culture was pipetted into a 1.5 ml centrifuge tube. This was centrifuged at 13,000 rpm for 10 minutes and supernatant was discarded. The pellet was resuspended in 100µl loading buffer. The samples were boiled with closed lids at 98°C for at least 10 minutes under the hood, after which they were let cool down and centrifuged at 13,000 rpm for another 10 minutes. 25µl were loaded in a 3-8% Tris-Acetate Gradient Gel from Invitrogen, 10µl NOVEX Pre-Stained Standard was used and PAGE was let run for 1 hour at 180 V with Tris-Acetate running buffer from Invitrogen. The aforementioned loading buffer is composed of 800µl ddH20, 250µl 0.5M Tris-HCl (pH = ~7.3), 400µl Glycerol (50%), 400µl SDS (10%), 100µl β-Mercaptoethanol (99%) and bromophenol-blue to obtain 2ml of loading buffer.

Software

Main packages

Python(2.7): The high-level programming language [http://www.python.org/ python] was used in combination with Django to develop the web-based interface and client-side applications.

Packages, plugins or libraries loaded by some of our python functions

Requests: The purpose of this library is to do simple http requests.

Biopython (>= 1.61): Just like bioperl this package contains tools for computational molecular biology.

xhtml2pdf: The converter package xhtml2pdf was applied to generate pdf from HTML/CSS templates.

BeautifulSoup: The BeautifulSoup HTML/XML parser was used for quick-turnaround applications.

celery: Asynchronous scheduling framework.

kombu: Database-based message passing framework.

python-openbabel(>= 2.3.2) : Python bindings to openbabel for 2D (chemical) structure generation.

UNAFold and MFold utils: UNAFold was required by Gibthon for DNA/RNA folding predictions.

MySQL: The relational database management system MySQL was utilized for database creation and maintenance.

MySQL - Python connector: A conncetor between the programming language python and the database management system.

Django(>= 1.5): The web application framework Django was used to achieve an easier database connection and is based on python. Since we improved and added basic functions of Gibthon (iGEM Team Cambridge 2010), the integration was easier to handle using the same frame work.

Django packages

django-registration: Handles user registrations and logins.

django-annoying: Gibthon uses this package. It is thought to eliminate annoying things in Django.

south: The south package handles database migration (database independent).

django-celery: The package was used for the integration of Celery (asynchronous task queue/job queue) into Django.

C++

CMake: Cross platform build system used by the NRPSDesigner.

MySQL C++ connector: C++ library for MySQL access.

libXML: C library for XML handling.

libcurl: C library for client-side URL transfers.

Boost.program_options: C++ library for command-line option parsing.

GCC (>= 4.8) or a comparable compiler with C++11 support

Javascript

Intro.js: This Javascript has been adopted for the guided step-by-step tour on the NRPS Designer homepage.

select2.js: The Select2 library is a jQuery based library for the replacements of select boxes, which support different connections to other functions.

@@ Line 209: / Line 209: @@
 ==Main packages==
-* '''Python(2.7)''': The high-level programming language python was used in combination with Django to develop the web-based interface and client-side applications.
+* '''Python(2.7)''': The high-level programming language [http://www.python.org/ python] was used in combination with Django to develop the web-based interface and client-side applications.
 ===Packages, plugins or libraries loaded by some of our python functions===

Team:Heidelberg/Templates/Methods