Difference between revisions of "Team:NTNU Trondheim/Experiments"

Revision as of 19:09, 15 September 2015

Jump to:
Cell Encapsulation
Alginate solution
Gelling Solution
Washing solution
Poly-L-lysine
Washing solution
LB medium
PBS medium
m-Toluic acid
Preparation of cell culture
Equipment needed
Preparation of alginate/cell mixture
Cell encapsulation
Capsule wash
Poly-L-lysine coating of capsules
Interlab
Preparation of samples
Acquisition of images by confocal microscopy
Quantitative image analysis
Molecular Genetics
LB media
LA media
Antibiotic stocks (according to iGEM protocol)
Making P. putida electrocompetent
Electroporation
Plating
Heat transformation of E.coli
Miniprep: Preparation of DNA from E.coli
Nanodrop: Measurement of DNA concentration
Enzyme digest (according to iGEM protocol)
Ligation (according to iGEM protocol)
Polymerase Chain Reaction (PCR)
Gelelectrophoresis
Spectrophotometer
Gel extraction
Flow Cytometry
PBS
HIFI
TE Buffer

Experiments & Protocols

Filter by category:
show all categories

"_"

Encapsulation

Interlab

Genetics

Modeling

only

Alginate solution

3.6 % (w/v) alginate in 0.5 % (w/v) NaCl
Stir until completely dissolved
Store in the fridge until the next day (to remove most of the air bubbles)

(The type of alginate used during this project is LF10/60 S12727 from FMC Biopolymer, Norway. Properties: FG = 0.65, FM = 0.35, MW = 113500 g/mol, [η] = 635)

Gelling solution

50 mM CaCl₂
1 mM BaCl₂
0.5 % (w/v) NaCl
Autoclave for 20 minutes at 120°C

Washing solution

2 mM CaCl2
0.5 % (w/v) NaCl
Autoclave for 20 minutes at 120°C

Poly-L-lysine

Poly-L-lysine in HBr: 8 mg in 10 ml of NaCl 0.5 % (w/v)
Poly-L-lysine in HCl: 10 mg in 10 ml of NaCl 0.5 % (w/v)

Washing solution (for poly-L-lysine-coated capsules)

0.3 % (w/v) mannitol in sterile water

LB medium for capsule leakage tests

2mM CaCl2 in standard LB + 35 μg/ml Kanamycin

PBS medium for capsule leakage tests

PBS 1X + 35 μg/ml Kanamycin

m-Toluic acid (inducer for P. putida pHH+GFP)

1 M in pure ethanol (working concentration 1 mM)

Preparation of cell culture

Overnight inoculation of P. putida

Inoculate P. putida from -80 ^oC freezer in 3 ml LB + 35 μg/ml Kanamycin
Incubate overnight at 30 ^oC

Equipment needed for generating alginate microcapsules

Electrostatic capsule generator (with a needle suitable for generating capsules of desired size)
Syringe feeder and disposable sterile syringes
Surgical tubing for connecting the needle to the syringe
Magnetic stirrer
Glassware

NOTE: Apart from the electrostatic capsule generator and the syringe feeder, all equipment and glassware should be sterilised prior to use.

Preparation of alginate/cell mixture

Centrifuge ON culture and pour off supernatant. Resuspend cells in PBS 1X
Adjust OD to 0.1
Carefully mix the alginate solution and cell culture 1:1 (the concentration of alginate in the mixture should be 1.8 % (w/v)), without incorporating air to the mixture
Place the desired volume (0.5 - 3.0 ml) into a disposable 5 ml syringe (BD Plastipak

Cell encapsulation

Connect the syringe containing the alginate/cell mixture to the needle by surgical tubing
Place the syringe in the syringe feeder, find the correct syringe type and dimensions in the instrument’s menu. Set syringe feeder speed.
Place the syringe feeder close to the electrostatic capsule generator, and place the needle in the needle holder
Pour gelling solution in a short and wide glass containing a magnet, and start the magnetic stirrer underneath
Adjust the needle holder’s distance to the gelling bath surface
Set a suitable voltage on the electrostatic capsule generator, and flick the start-switch
Wait until all of the alginate/cell mixture has passed through the needle

(For generating capsules with a diameter of ~230 μm the following parameters were chosen: inner diameter of needle 130 μm, speed 6 ml/hour, voltage 5000 V, distance between needle and gelling bath 1 - 2 cm)

Capsule wash

Let the capsules stay in the gelling bath for a few minutes
Gently pour the gelling solution containing the capsules through a filter (mesh, pore size 40 μm), while holding the filter holder tube at an angle
Put the filter holder containing the capsules in an unused and sterile beaker, gently flush the capsules with the washing solution (mannitol solution if coating the capsules in poly-L-lysine) using a 25 mL graduated pipette (repeat 2 times)
Put the filter holder containing the capsules in an unused and sterile beaker, gently resuspend the capsules in the desired volume of storage medium or continue with coating the capsules in poly-L-lysine

Poly-L-lysine coating of capsules

Prepare fresh poly-L-lysine solution
Following the cell encapsulation, wash the capsules in mannitol (as described under the capsule wash protocol)
Transfer the capsules to a poly-L-lysine bath (with magnetic stirrer)
Wait 10 minutes before filtering the capsules (as described under the capsule wash protocol)
Gently pour the poly-L-lysine solution containing the capsules through a filter (mesh, pore size 40 μm), while holding the filter holder tube at an angle
Put the filter holder containing the capsules in an unused and sterile beaker, gently flush the capsules with 0.5 % (w/v) NaCl solution using a 25 mL graduated pipette (repeat 2 times)
Put the filter holder containing the capsules in an unused and sterile beaker, gently resuspend the capsules in the desired volume of storage medium

Preparation of samples

Overnight inoculation at 37oC of each biological replicate of devices 1-3 + positive and negative controls in 3 ml LB + Kanamycin (Chloramphenicol for the controls)
5 % inoculation at 37oC in 3 ml LB + Kanamycin (Chloramphenicol for the controls)
Measure OD, and dilute/adjust OD to 0.3 (+/- 0.025). This was found (by trial and error) to be a suitable density of cells for confocal microscopy.
Cell cultures were then put on ice and transported to the microscope.
For each biological replicate, 1.5 μl of the sample was put on a rectangular cover glass of thickness 1.5 (served as a suitable object glass for confocal microscopy). A small square-shaped cover glass of thickness 1.5 was put on top of the sample. To reduce bacteria moving and being pulled towards the edges of the cover glass, its edges were sealed with nail polish.
For each prepared microscopy sample (biological replicate), three images were obtained to serve as technical replicates. These three images should be taken at different xy positions in the prepared microscopy sample.

NOTE: The small volume of 1.5 μl ensures a short enough distance between the glasses (to work well with the z section size set by the pinhole size, see below), so that all bacteria between the glasses in a given xy(z) frame can be in focus and thus their fluorescence be detected by the microscope.

Acquisition of images by confocal microscopy

A Leica SP5 confocal microscope was used with the following parameters set in the LAS AF software: Argon laser (power 20 %) excitation line 488 nm (set to 6%), hybrid detector (HyD, Leica) set to filter and detect emitted light between 495-600 nm, pinhole size 399.99 nm (with a corresponding z section thickness of 2.897 μm).
The objective used was HCX PL APO CS (63x magnification, water immersion, numerical aperture 1.2, XY resolution 163 and Z resolution 290 at 488 nm).
Look into the microscope to focus in on your bacteria (bright field).
For live view, set the format to 512 * 512 and speed to 400 Hz, and press ‘Live’.
Adjust the focus with the z-position wheel, and when a good view of the bacteria and their fluorescence has been found in live view, stop the live view to scan the image. The following parameters were set in the LAS AF software to acquire the images: bit-depth of 12 (found under the configuration tab), acquisition mode xyz, zoom 1.25, speed 100 Hz, format 2048 * 2048 (pixel size 96.15 nm * 96.15 nm, image size 196.83 μm * 196.83 μm), line average 3.
The Leica SP5 should be set to acquire both a fluorescence image (with the HyD detector) and a regular bright field image (it is important to set both channels to ‘Visible’). The settings for the bright field channel were Smart Gain 305 V, Smart Offset -2.5 %. The setting for the fluorescence channel was Smart Gain 10.0 % (the HyD detector sets the offset by itself).
Each captured image (containing TIFF files of both bright field and fluorescence images) should be saved automatically under the same experiment (LIF file). This enables the microscopist to export the LIF file, which is then ready for the subsequent analysis.

NOTE: A bit-depth of 12 was chosen in this Interlab study to ensure that the range would accommodate both the samples with strong fluorescence and weak fluorescence. A lower bit-depth like 8 bit would cause the images with strong fluorescence to be oversaturated because the range would simply not be wide enough (data lost before analysis).

Quantitative image analysis

The data set was provided by the microscope software in LIF format. This file has about 1GB size. While ImageJ can read LIF format, it does not manage the memory very well, and cannot load the images simultaneously. The method used to circumvent this issue is to convert the LIF file into many TIFF files. Each TIFF file contained one fluorescence image and one bright field image. bfconvert utility was downloaded to convert the Leica LIF files into a set of TIFF files. The utility was downloaded from http://downloads.openmicroscopy.org/bio-formats/5.1.3/.

The images were converted with the command line:

$ ./bfconvert 150818\ Interlab\ Hyd2.lif %n.tiff

The resulting files have spaces in their name. This created problems for ImageJ. The spaces were converted into underscores with the following command:
```
$ for file in *.tiff; do mv "$file" `echo $file | tr ' ' '_'` ; done
```
The ImageJ analysis was scripted into a macro file. The commands of the macro file are as described in the following instructions.
The TIFF image was opened with ImageJ.
The two stacks of the images were converted into two images for easy reference in the macro.
The bright field image was selected and viewed with a Grays LUT.
The background was subtracted using Process > Subtract background...". In the new window, the rolling ball radius is 50 px, and the "Light" was ticked. A new window appeared asking whether to process the images. "No" was clicked.
The threshold was determined by clicking Image > Adjust > Threshold… "Apply" was clicked. On the new window, Background: Light was selected. Calculate threshold for each image was unticked. "Apply" was clicked/.
Analyze > Set Measurements was selected, "Area", and "Mean" are ticked, and Redirect to: was set the name of the first image (fluorescence).
Analyze > Analyze Particles was clicked. For size, 2E-8 was set as the lower bound. For circularity, 0.10 to 0.7 was set, and show outline was selected for visualization purposes.
Results window was selected, and saved as a CSV file.
In MATLAB, all the batch processed files were converted into a 3D matrix TABLE (Devices, Biological replicates, Technical replicates), the intensity was calculated as MEAN(TABLE,3), which is the average over the technical replicates. These results were satisfactorily compared with the flow cytometry results.

LB media

10% Tryptone
5% Yeast extract
5% NaCl

Autoclave for 20 minutes at 120°C
Add appropriate antibiotics up to respective working concentration

LA media

10% Tryptone
5% Yeast extract
5% NaCl
20% Agar
Autoclave for 20 minutes at 120°C
Add appropriate antibiotics up to respective working concentration

Antibiotic stocks (according to iGEM protocol)

Ampicillin: 100mg/ml in 50% EtOH
Chloramphenicol: 35mg/ml in 100% EtOH
Kanamycin: 35mg/ml in distilled H20
Aliquots of 500ul, stored at -20°C

Making P. putida electrocompetent

Inoculate of 1% from ON culture in 10ml LB media
Incubate at 30°C until OD600=0.45+-10%
Aliquot 1.5ml cell suspension in an eppendorf tube per electroporation
Centrifugate at 12000g for 1 minute at 4°C
Remove supernatant
Add 1ml 300mM sucrose (cold)
Centrifugate at 12000g for 1 minute at 4°C
Remove supernatant
Add 50ul 300mM sucrose (cold)
Keep on ice until used for electroporation

Electroporation

Add plasmid (1-5ul, up to 500ng DNA) to 50ul electrocompetent cells into electroporation
Electroporation with Biorad GenePulser Xcell

Voltage: 2500V
Capacitance: 25uF
Resistence: 200(Ohm)
Cuvette: 2mm)

Add 1ml LB media to the electroporation chamber immediately after pulse
Transfer all liquid from the electroporation chamber into an eppendorf tube
Incubate for 1 hour at 30°C
Plate cells

Plating

Transfer 50ul of cell suspension in the middle of the LA plate (with appropriate antibiotic)
Distribute cells equally over the whole plate
Centrifuge remaining cell suspension and remove supernatant until less then 100ul remains
Resuspend cells in remaining supernatant
Transfer all remaining cell suspension onto a second LA plate
Distribute cells equally over the whole plate

Heat transformation of E.coli

Add plasmid (1-5ul, up to 500ng DNA) to 100ul competent cells (stored at -80°C) in eppendorf tube
Keep on ice for 30 minutes
Keep in water bath for 45 seconds at 42°C
Keep on ice for 3 minutes
Add 500ul LB media
Incubate for 1 hour at 37°C

Miniprep: Preparation of DNA from E.coli

According to Wizard Plus SV Minipreps DNA Purification system

Nanodrop: Measurement of DNA concentration

Enzyme digest (according to iGEM protocol)

Keep all enzymes and buffers used on ice
Add 250ng of DNA to 3ul of Cutsmart Buffer and 0.5ul of each appropriate enzyme. Fill up with nuclease free water to a total volume of 16ul
Incubate mixture for 30 minutes at 37°C (enzyme digest)
Incubate mixture for 20 minutes at 80°C (heat inactivation)

Ligation (according to iGEM protocol)

Add 25ng of DNA to 1ul T4 DNA Ligase buffer and 0.5ul T4 DNA Ligase. Fill up with nuclease free water to a total colume of 10ul
Incubate for 30 minutes at 16°C (ligation) *or overnight
Incubate for 20 minutes at 65°C (heat inactivation)
Transformation with 2ul of product

Polymerase Chain Reaction (PCR)

Recipe

2.5ul forward primer
2.5ul reverse primer
1ul dNTP
<1000ng template DNA
0.5ul Q5 polymerase
10ul Q5 buffer
10ul High GC enhancer
Fill up with nuclease free water to a total volume of 50ul

Protocol

30 seconds - 98°C
10 seconds - 98°C
20 seconds - Annealing temperature
80 seconds - 72°C
Repeat steps 2.-4. 20-30 times
2 minutes - 72°C
Infinite hold at 4°C

Gelelectrophoresis

Spectrophotometer

Gel extraction

Flow Cytometry

Inoculate 1% of overnight culture in 3ml LB media
Incubate at the appropriate temperature (30°C for P.putida, 37°C for E.coli)
Dilute 1:100 in PBS, total volume: 1.5ml
Transfer 500ul in flow cytometry tube (per technical replicate)
Flow cytometry with BD Accuri C6 sampler

Suction: 2 minutes
Flow rate: 35ul/minute
Pre-set thresholds: FSC=4000, FL-1=200
Washing steps after last technical replicate from each sample

@@ Line 9: / Line 9: @@
 				<!-- -------------------Month Filters------------------- -->
 				<li><h6> Jump to: </h6></li>
-				<!--li class="nb-month">
-					<div style="top:67px;margin-left:-6px">Spring</div>
-				</li>
-				<li id="weekA" class="active" onclick="weekFilter(this)">
-					<a class="nb-week" href="#A">January</a>
-				</li>
-				<li id="weekB" onclick="weekFilter(this)">
-					<a class="nb-week" href="#B">February</a>
-				</li>
-				<li id="weekC" onclick="weekFilter(this)">
-					<a class="nb-week" href="#C">March</a>
-				</li>
-				<li id="weekD" onclick="weekFilter(this)">
-					<a class="nb-week" href="#D">April</a>
-				</li>
-				<li id="weekE" onclick="weekFilter(this)">
-					<a class="nb-week" href="#E">May</a>
-				</li-->
 				<li class="nb-month">
-					<div style="top:55px">Media</div>
+					<div style="top:200px;margin-left:-50px">Cell Encapsulation</div>
-				</li>
-				<li id="week1" onclick="weekFilter(this)">
-					<a class="nb-week" href="#1">Lysogeny Broth (LB)</a>
-				</li>
-				<li id="week2" onclick="weekFilter(this)">
-					<a class="nb-week" href="#2">SOC medium</a>
-				</li>
-				<li id="week3" onclick="weekFilter(this)">
-					<a class="nb-week" href="#3">yB medium</a>
-				</li>
-				<li id="week4" onclick="weekFilter(this)">
-					<a class="nb-week" href="#4"><i>Synechocystis</i> medium</a>
-				</li>
-				<li id="week5" onclick="weekFilter(this)">
-					<a class="nb-week" href="#5"></a>
 				</li>
+			    <li id="week1" onclick="weekFilter(this)">
+			    	<a class="nb-week" href="#1">Alginate solution</a>
+			    </li>
+			    <li id="week2" onclick="weekFilter(this)">
+			    	<a class="nb-week" href="#2">Gelling Solution</a>
+			    </li>
+			    <li id="week3" onclick="weekFilter(this)">
+			    	<a class="nb-week" href="#3">Washing solution</a>
+			    </li>
+			    <li id="week4" onclick="weekFilter(this)">
+			    	<a class="nb-week" href="#4">Poly-L-lysine</a>
+			    </li>
+			    <li id="week5" onclick="weekFilter(this)">
+			    	<a class="nb-week" href="#5">Washing solution</a>
+			    </li>
+			    <li id="week6" onclick="weekFilter(this)">
+			    	<a class="nb-week" href="#6">LB medium</a>
+			    </li>
+			    <li id="week7" onclick="weekFilter(this)">
+			    	<a class="nb-week" href="#7">PBS medium</a>
+			    </li>
+			    <li id="week8" onclick="weekFilter(this)">
+			    	<a class="nb-week" href="#8">m-Toluic acid</a>
+			    </li>
+			    <li id="week9" onclick="weekFilter(this)">
+			    	<a class="nb-week" href="#9">Preparation of cell culture</a>
+			    </li>
+		        <li id="week10" onclick="weekFilter(this)">
+		            <a class="nb-week" href="#10">Equipment needed</a>
+		        </li>
+		        <li id="week11" onclick="weekFilter(this)">
+		            <a class="nb-week" href="#11">Preparation of alginate/cell mixture</a>
+		        </li>
+		        <li id="week12" onclick="weekFilter(this)">
+		            <a class="nb-week" href="#12">Cell encapsulation</a>
+		        </li>
+		        <li id="week13" onclick="weekFilter(this)">
+		            <a class="nb-week" href="#13">Capsule wash</a>
+		        </li>
+		        <li id="week14" onclick="weekFilter(this)">
+		            <a class="nb-week" href="#14">Poly-L-lysine coating of capsules</a>
+		        </li>
 				<li class="nb-month">
-					<div style="top:140px;margin-left:-18px">Techniques</div>
+					<div style="top:40px;margin-left:-12px">Interlab</div>
-				</li>
-				<li id="week6" onclick="weekFilter(this)">
-					<a class="nb-week" href="#6">Gibson assembly</a>
-				</li>
-				<li id="week7" onclick="weekFilter(this)">
-					<a class="nb-week" href="#7">DNA isolation and cleaning</a>
-				</li>
-				<li id="week8" onclick="weekFilter(this)">
-					<a class="nb-week" href="#8">DNA digestion</a>
-				</li>
-				<li id="week9" onclick="weekFilter(this)">
-					<a class="nb-week" href="#9">PCR</a>
-				</li>
-				<li id="week10" onclick="weekFilter(this)">
-					<a class="nb-week" href="#10">NanoDrop</a>
-				</li>
-				<li id="week11" onclick="weekFilter(this)">
-					<a class="nb-week" href="#11">3A assembly</a>
-				</li>
-				<li id="week12" onclick="weekFilter(this)">
-					<a class="nb-week" href="#12">Ligation</a>
-				</li>
-				<li id="week13" onclick="weekFilter(this)">
-					<a class="nb-week" href="#13">Transformation (<i>Escherichia coli</i>)</a>
-				</li>
-				<li id="week14" onclick="weekFilter(this)">
-					<a class="nb-week" href="#14">Transformation (<i>Synechocystis sp. PCC 6803</i>)</a>
 				</li>
+		        <li id="week15" onclick="weekFilter(this)">
+		            <a class="nb-week" href="#15">Preparation of samples</a>
+		        </li>
+		        <li id="week16" onclick="weekFilter(this)">
+		            <a class="nb-week" href="#16">Acquisition of images by confocal microscopy</a>
+		        </li>
+		        <li id="week17" onclick="weekFilter(this)">
+		            <a class="nb-week" href="#17">Quantitative image analysis</a>
+		        </li>
 				<li class="nb-month">
-					<div style="top:52px;margin-left:-9px">Plasmids</div>
+					<div style="top:260px;margin-left:-50px">Molecular Genetics</div>
 				</li>
-				<li id="week16" onclick="weekFilter(this)">
-					<a class="nb-week" href="#16">Right flank</a>
+		        <li id="week18" onclick="weekFilter(this)">
-				</li>
+		            <a class="nb-week" href="#18">LB media</a>
-				<li id="week17" onclick="weekFilter(this)">
+		        </li>
-					<a class="nb-week" href="#17">Left flank</a>
-				</li>
-				<li id="week18" onclick="weekFilter(this)">
-					<a class="nb-week" href="#18">Kanamycin resistance</a>
-				</li>
-				<li id="week19" onclick="weekFilter(this)">
-					<a class="nb-week" href="#19">Lac promoter</a>
-				</li>
-				<li id="week20" onclick="weekFilter(this)">
-					<a class="nb-week" href="#20">Glucose oxidase</a>
-				</li>
-								<li id="week21" onclick="weekFilter(this)">
-					<a class="nb-week" href="#21">Left flank + Kan + Right flank</a>
-				</li>
-				<li class="nb-month">
-					<div style="top:55px;margin-left:-29px">Calculations</div>
+		        <li id="week19" onclick="weekFilter(this)">
-				</li>
+		            <a class="nb-week" href="#19">LA media</a>
-				<li id="week24" onclick="weekFilter(this)">
+		        </li>
-					<a class="nb-week" href="#24">DNA concentration</a>
-				</li>
+		        <li id="week20" onclick="weekFilter(this)">
+		            <a class="nb-week" href="#20">Antibiotic stocks (according to iGEM protocol)</a>
+		        </li>
+		        <li id="week21" onclick="weekFilter(this)">
+		            <a class="nb-week" href="#21">Making P. putida electrocompetent</a>
+		        </li>
+		        <li id="week22" onclick="weekFilter(this)">
+		            <a class="nb-week" href="#22">Electroporation</a>
+		        </li>
+		        <li id="week23" onclick="weekFilter(this)">
+		            <a class="nb-week" href="#23">Plating</a>
+		        </li>
+		        <li id="week24" onclick="weekFilter(this)">
+		            <a class="nb-week" href="#24">Heat transformation of E.coli </a>
+		        </li>
+		        <li id="week25" onclick="weekFilter(this)">
+		            <a class="nb-week" href="#25">Miniprep: Preparation of DNA from E.coli</a>
+		        </li>
+		        <li id="week26" onclick="weekFilter(this)">
+		            <a class="nb-week" href="#26">Nanodrop: Measurement of DNA concentration</a>
+		        </li>
+		        <li id="week27" onclick="weekFilter(this)">
+		            <a class="nb-week" href="#27">Enzyme digest (according to iGEM protocol)</a>
+		        </li>
+		        <li id="week28" onclick="weekFilter(this)">
+		            <a class="nb-week" href="#28">Ligation (according to iGEM protocol)</a>
+		        </li>
+		        <li id="week29" onclick="weekFilter(this)">
+		            <a class="nb-week" href="#29">Polymerase Chain Reaction (PCR)</a>
+		        </li>
+		        <li id="week30" onclick="weekFilter(this)">
+		            <a class="nb-week" href="#30">Gelelectrophoresis</a>
+		        </li>
+		        <li id="week31" onclick="weekFilter(this)">
+		            <a class="nb-week" href="#31">Spectrophotometer</a>
+		        </li>
+		        <li id="week32" onclick="weekFilter(this)">
+		            <a class="nb-week" href="#32">Gel extraction</a>
+		        </li>
+		        <li id="week33" onclick="weekFilter(this)">
+		            <a class="nb-week" href="#33">Flow Cytometry</a>
+		        </li>
+		        <li id="week34" onclick="weekFilter(this)">
+		            <a class="nb-week" href="#34">PBS</a>
+		        </li>
+		        <li id="week35" onclick="weekFilter(this)">
+		            <a class="nb-week" href="#35">HIFI</a>
+		        </li>
+		        <li id="week36" onclick="weekFilter(this)">
+		            <a class="nb-week" href="#36">TE Buffer</a>
+		        </li>
 			</ul>
 		</div>
@@ Line 120: / Line 180: @@
 			<div class="row last-ele nb-labels" style="padding-bottom:0px">
 				<div style="opacity:0.00;width:0px">"_"</div>
-				<div>Media</div>
+				<div>Encapsulation</div>
-				<div>Techniques</div>
+				<div>Interlab</div>
-				<div>Plasmids</div>
+				<div>Genetics</div>
-				<div>Calculations</div>
+				<div>Modeling</div>
 			</div>
 			<div class="row">
 	<!-- PLACEHOLDER -->    <div class="two columns nb-filter" style="opacity:0.00;width:0px">
+				</div>
+				<div class="two columns nb-filter">
+					<div class="filter-container" onclick="filter(this)" style="background-image:url(https://static.igem.org/mediawiki/2015/1/19/NTNU_Trondheim_techniques_off.png)">
+						<img id="techniques" src=" https://static.igem.org/mediawiki/2015/b/b2/NTNU_Trondheim_techniques_on.png">
+					</div>
+					<div id="techniques-only" class="nb-only" onclick="onlyFilter(this)">only</div>
 				</div>
 				<div class="two columns nb-filter">
@@ Line 133: / Line 200: @@
 					</div>
 					<div id="media-only" class="nb-only" onclick="onlyFilter(this)">only</div>
-				</div>
-				<div class="two columns nb-filter">
-					<div class="filter-container" onclick="filter(this)" style="background-image:url(https://static.igem.org/mediawiki/2015/1/19/NTNU_Trondheim_techniques_off.png)">
-						<img id="techniques" src=" https://static.igem.org/mediawiki/2015/b/b2/NTNU_Trondheim_techniques_on.png">
-					</div>
-					<div id="techniques-only" class="nb-only" onclick="onlyFilter(this)">only</div>
 				</div>
 				<div class="two columns nb-filter">
@@ Line 156: / Line 217: @@
 			</div>
 				<!-- -------------------Protocol entries------------------- -->
-			<div id="week1entry" class="nb-week" style="display: block">
-				<div class="twelve columns">
-					<h3 class="centered">Lysogeny Broth (LB)</h3>
-</p>
+				<!-- Begin Entry -->
+				<div id="week1entry" class="nb-week" style="display: block">
+					<div class="twelve columns">
+						<div class="entry pc-techniques">
+							<div>
+								<h3 class="centered">
+									Alginate solution
+								</h3>
+								<ul>
+								<li>
+.6 % (w/v) alginate in 0.5 % (w/v) NaCl
+								</li>
+								<li>
+									Stir until completely dissolved
+								</li>
+								<li>
+									Store in the fridge until the next day (to remove most of the air bubbles)
+								</li>
+								</ul>
+								<p>(The type of alginate used during this project is LF10/60 S12727 from FMC Biopolymer, Norway. Properties: FG = 0.65, FM = 0.35, MW = 113500 g/mol, [η] = 635)</p>
+							</div>
+						</div>
+					</div>
+				</div>
+				<!-- End Entry -->
-<div class="entry pc-media">
+				<!-- Begin Entry -->
-	<div>
+				<div id="week2entry" class="nb-week" style="display: block">
-		<h6>Recipe
+					<div class="twelve columns">
-			<div class="nb-onetech-i nohilite">Antibiotic additions</div>
+						<div class="entry pc-techniques">
-		</h6>
+							<div>
-		<div class="nb-tech">
+								<h3 class="centered">
-			<div class="AB-table">
+								Gelling solution
-				<table>
+								</h3>
-					<tr>
+								<ul>
-						<th> Antibiotic</th> <th> Stock concentration</th> <th>Final concentration</th> <th>Dillution factor</th> <th>Solvent</th> <th>Storage temperature</th>
+								<li>
- 					</tr>
+mM CaCl<sub>2</sub>
- 					<tr>
+								</li>
- 						<td>Ampicillin</td> <td>50 mg / mL</td> <td>50 &mu;g / mL</td> <td>1000</td> <td>Filter sterilized H<sub>2</sub>O</td> <td> 4 &deg;C</td>
+								<li>
- 					</tr>
+mM BaCl<sub>2</sub>
- 					<tr>
+								</li>
- 						<td>Chloramphenicol</td><td>30 mg / mL</td><td>30 &mu;g / mL</td> <td>1000</td><td>Ethanol</td><td>-20 &deg;C</td>
+								<li>
- 					</tr>
+.5 % (w/v) NaCl
- 					<tr>
+								</li>
- 						<td>Kanamycin</td><td>50 mg / mL</td><td>30 &mu;g / mL</td><td>1000</td><td>Filter sterilized H<sub>2</sub>O</td><td>4 &deg;C</td>
+								<li>
- 					</tr>
+								Autoclave for 20 minutes at 120°C
- 					<tr>
+								</li>
- 						<td>Spectinomycin</td><td>50 mg / mL</td><td>50 &mu;g / mL</td><td>1000</td><td>Filter sterilized H<sub>2</sub>O</td><td>4 &deg;C</td>
+								</li>
- 					</tr>
+								</ul>
-				</table>
+							</div>
-			</div>
+						</div>
-		</div>
+					</div>
-			<p>
+				</div>
-				Ingredients: <br> <ul> <li>Tryptone (10g)</li>  <li>NaCl (10g)</li> <li>Yeast Extract (5g)</li> </ul> <br> <p> <ol> <li>Fill with 1 L of distilled / filtered H<sub>2</sub>O.</li> <li>Autoclave at 121 &deg;C for 20 minutes. </li> <li>Add antibiotics if needed, after the medium has cooled down.</li>
+				<!-- End Entry -->
-				</ol></p>
-			</p>
+				<!-- Begin Entry -->
-	</div>
+				<div id="week3entry" class="nb-week" style="display: block">
-</div>
+					<div class="twelve columns">
+						<div class="entry pc-techniques">
+							<div>
+								<h3 class="centered">
+								Washing solution
+								</h3>
+								<ul>
+								<li>
+mM CaCl2
+								</li>
+								<li>
+.5 % (w/v) NaCl
+								</li>
+								<li>
+								Autoclave for 20 minutes at 120°C
+								</li>
+								</ul>
+							</div>
+						</div>
+					</div>
+				</div>
+				<!-- End Entry -->
+				<!-- Begin Entry -->
+				<div id="week4entry" class="nb-week" style="display: block">
+					<div class="twelve columns">
+						<div class="entry pc-techniques">
+							<div>
+								<h3 class="centered">
+								Poly-L-lysine
+								</h3>
+								<ul>
+								<li>
+								Poly-L-lysine in HBr: 8 mg in 10 ml of NaCl 0.5 % (w/v)
+								</li>
+								<li>
+								Poly-L-lysine in HCl: 10 mg in 10 ml of NaCl 0.5 % (w/v)
+								</li>
+								</ul>
+							</div>
+						</div>
+					</div>
+				</div>
+				<!-- End Entry -->
+				<!-- Begin Entry -->
+				<div id="week5entry" class="nb-week" style="display: block">
+					<div class="twelve columns">
+						<div class="entry pc-techniques">
+							<div>
+								<h3 class="centered">
+								Washing solution (for poly-L-lysine-coated capsules)
+								</h3>
+								<ul>
+								<li>
+.3 % (w/v) mannitol in sterile water
+								</li>
+								</ul>
+							</div>
+						</div>
+					</div>
+				</div>
+				<!-- End Entry -->
+				<!-- Begin Entry -->
+				<div id="week6entry" class="nb-week" style="display: block">
+					<div class="twelve columns">
+						<div class="entry pc-techniques">
+							<div>
+								<h3 class="centered">
+								LB medium for capsule leakage tests
+								</h3>
+								<ul>
+								<li>
+mM CaCl2 in standard LB + 35 μg/ml Kanamycin
+								</li>
+								</ul>
+							</div>
+						</div>
+					</div>
+				</div>
+				<!-- End Entry -->
+				<!-- Begin Entry -->
+				<div id="week7entry" class="nb-week" style="display: block">
+					<div class="twelve columns">
+						<div class="entry pc-techniques">
+							<div>
+								<h3 class="centered">
+								PBS medium for capsule leakage tests
+								</h3>
+								<ul>
+								<li>
+								PBS 1X + 35 μg/ml Kanamycin
+								</li>
+								</ul>
+							</div>
+						</div>
+					</div>
+				</div>
+				<!-- End Entry -->
+				<!-- Begin Entry -->
+				<div id="week8entry" class="nb-week" style="display: block">
+					<div class="twelve columns">
+						<div class="entry pc-techniques">
+							<div>
+								<h3 class="centered">
+								m-Toluic acid (inducer for <i>P. putida</i> pHH+GFP)
+								</h3>
+								<ul>
+								<li>
+M in pure ethanol	(working concentration 1 mM)
+								</li>
+								</ul>
+							</div>
+						</div>
+					</div>
+				</div>
+				<!-- End Entry -->
+				<!-- SECTION: CELL ENCAPSULATION -->
+				<!-- Begin Entry -->
+				<div id="week9entry" class="nb-week" style="display: block">
+					<div class="twelve columns">
+						<div class="entry pc-techniques">
+							<div>
+								<h3 class="centered">
+									Preparation of cell culture
+								</h3>
+								<p>Overnight inoculation of P. putida</p>
+								<ul>
+								<li>
+								Inoculate P. putida from -80 <sup>o</sup>C freezer in 3 ml LB + 35 μg/ml Kanamycin
+								</li>
+								<li>
+								Incubate overnight at 30 <sup>o</sup>C
+								</li>
+								</ul>
+							</div>
+						</div>
+					</div>
+				</div>
+				<!-- End Entry -->
+                <!-- Begin Entry -->
+				<div id="week10entry" class="nb-week" style="display: block">
+					<div class="twelve columns">
+						<div class="entry pc-techniques">
+							<div>
+        						<h3 class="centered">
+    							Equipment needed for generating alginate microcapsules
+        						</h3>
+								<ul>
+									<li>
+										Electrostatic capsule generator (with a needle suitable for generating capsules of desired size)
+									</li>
+									<li>
+										Syringe feeder and disposable sterile syringes
+									</li>
+									<li>
+										Surgical tubing for connecting the needle to the syringe
+									</li>
+									<li>
+										Magnetic stirrer
+									</li>
+									<li>
+										Glassware
+									</li>
+								</ul>
+								<b>NOTE</b>: Apart from the electrostatic capsule generator and the syringe feeder, all equipment and glassware should be sterilised prior to use.
+							</div>
+						</div>
+					</div>
+				</div>
+				<!-- End Entry -->
-<p>
+                <!-- Begin Entry -->
-				</div>
+				<div id="week11entry" class="nb-week" style="display: block">
-			</div>
+					<div class="twelve columns">
+						<div class="entry pc-techniques">
+							<div>
+        						<h3 class="centered">
+    							Preparation of alginate/cell mixture
+        						</h3>
+								<ol>
-			<div id="week2entry" class="nb-week" style="display: block">
+									<li>
-				<div class="twelve columns">
+										Centrifuge ON culture and pour off supernatant. Resuspend cells in PBS 1X
-					<h3 class="centered">SOC medium</h3>
+									</li>
-</p>
+									<li>
-<div class="entry pc-media">
+										Adjust OD to 0.1
-	<div>
+									</li>
-		<h6>Recipe
+									<li>
-			<div class="nb-onetech-i nohilite">show technical details</div>
+										Carefully mix the alginate solution and cell culture 1:1 (the concentration of alginate in the mixture should be 1.8 % (w/v)), without incorporating air to the mixture
-		</h6>
+									</li>
-		<div class="nb-tech">{{{tech}}}</div>
+									<li>
-<p>		<p>		Ingredients (1L): <br> <ul> <li>Bactotryptone (20 g)</li>  <li>Yeast extract (5g)</li> <li>NaCl (0.584g)</li> <li>KCl (0.186g)</li> <li>Agar (20g ONLY IF MAKING PLATES)</li> </ul> <br> <p> <ol> <li>Fill with 0.5 L of distilled / filtered H<sub>2</sub>O.</li> <li>Autoclave at 121 &deg;C for 20 minutes. </li>  <li>Add 10 mL 1M MgCl<sub>2</sub>, 10 mL MgSO<sub>4</sub> and 20 mL 1M glucose (all should be sterile) prior to use </li>
+										Place the desired volume (0.5 - 3.0 ml) into a disposable 5 ml syringe (BD Plastipak
-				</ol>
+									</li>
-</p>
+								</ol>
-</p>
+							</div>
-	</div>
+						</div>
-</div>
+					</div>
+				</div>
+				<!-- End Entry -->
-</div>
+                <!-- Begin Entry -->
-</div>
+				<div id="week12entry" class="nb-week" style="display: block">
+					<div class="twelve columns">
+						<div class="entry pc-techniques">
+							<div>
+        						<h3 class="centered">
+    							Cell encapsulation
+        						</h3>
+								<ol>
+									<li>
+										Connect the syringe containing the alginate/cell mixture to the needle by surgical tubing
+									</li>
+									<li>
+										Place the syringe in the syringe feeder, find the correct syringe type and dimensions in the instrument’s menu. Set syringe feeder speed.
+									</li>
+									<li>
+										Place the syringe feeder close to the electrostatic capsule generator, and place the needle in the needle holder
+									</li>
+									<li>
+										Pour gelling solution in a short and wide glass containing a magnet, and start the magnetic stirrer underneath
+									</li>
+									<li>
+										Adjust the needle holder’s distance to the gelling bath surface
+									</li>
+									<li>
+										Set a suitable voltage on the electrostatic capsule generator, and flick the start-switch
+									</li>
+									<li>
+										Wait until all of the alginate/cell mixture has passed through the needle
+									</li>
+								</ol>
+								<p>(For generating capsules with a diameter of ~230 μm the following parameters were chosen: inner diameter of needle 130 μm, speed 6 ml/hour, voltage 5000 V, distance between needle and gelling bath 1 - 2 cm)</p>
+							</div>
+						</div>
+					</div>
+				</div>
+				<!-- End Entry -->
+                <!-- Begin Entry -->
+				<div id="week13entry" class="nb-week" style="display: block">
+					<div class="twelve columns">
+						<div class="entry pc-techniques">
+							<div>
+        						<h3 class="centered">
+    							Capsule wash
+        						</h3>
+								<ol>
-<div id="week3entry" class="nb-week" style="display: block">
+									<li>
-				<div class="twelve columns">
+										Let the capsules stay in the gelling bath for a few minutes
-					<h3 class="centered">yB medium</h3>
+									</li>
-</p>
+									<li>
-<div class="entry pc-media">
+										Gently pour the gelling solution containing the capsules through a filter (mesh, pore size 40 μm), while holding the filter holder tube at an angle
-	<div>
+									</li>
-		<h6>Recipe
+									<li>
+										Put the filter holder containing the capsules in an unused and sterile beaker, gently flush the capsules with the washing solution (mannitol solution if coating the capsules in poly-L-lysine) using a 25 mL graduated pipette (repeat 2 times)
-		</h6>
+									</li>
-		<div class="nb-tech">{{{tech}}}</div>
+									<li>
-<p>		Ingredients: <br> <ul> <li>Yeast extract (2.5g)</li>  <li>Bactotryptone (10g)</li> <li>KCL (0.38g)</li> </ul> <br> <p> <ol> <li>Fill with 0.5 L of distilled / filtered H<sub>2</sub>O.</li> <li>Add KOH until the pH is 7.4, then autoclave at 121 &deg;C for 20 minutes. </li>  <li>Add 17 mL sterile 1M MgSO4 </li>
+										Put the filter holder containing the capsules in an unused and sterile beaker, gently resuspend the capsules in the desired volume of storage medium or continue with coating the capsules in poly-L-lysine
-				</ol>
+									</li>
-</p>
+								</ol>
-</p>
+							</div>
-	</div>
+						</div>
-</div>
+					</div>
+				</div>
+				<!-- End Entry -->
-</div>
+                <!-- Begin Entry -->
-</div>
+				<div id="week14entry" class="nb-week" style="display: block">
+					<div class="twelve columns">
+						<div class="entry pc-techniques">
+							<div>
+        						<h3 class="centered">
+    							Poly-L-lysine coating of capsules
+        						</h3>
+								<ol>
-<div id="week4entry" class="nb-week" style="display: block">
+									<li>
-				<div class="twelve columns">
+										Prepare fresh poly-L-lysine solution
-					<h3 class="centered">Synechocystis medium</h3>
+									</li>
-</p>
+									<li>
-<div class="entry pc-media">
+										Following the cell encapsulation, wash the capsules in mannitol (as described under the capsule wash protocol)
-	<div>
+									</li>
-		<h6>Recipe
+									<li>
+										Transfer the capsules to a poly-L-lysine bath (with magnetic stirrer)
-		</h6>
+									</li>
-		<div class="nb-tech">{{{tech}}}</div>
+									<li>
-<p>		To grow Synechocystis cultures, BG-11 medium was made according to the recipe found on the <a href="http://unitedscientists.org/labs/norway/NTNU/PhotoSynLab/wiki/table-of-contents-2/protocol-physiological/bg-11-media">PhotoSynLab wiki</a>.
+										Wait 10 minutes before filtering the capsules (as described under the capsule wash protocol)
-</p>
+									</li>
-	</div>
+									<li>
-</div>
+										Gently pour the poly-L-lysine solution containing the capsules through a filter (mesh, pore size 40 μm), while holding the filter holder tube at an angle
+									</li>
+									<li>
+										Put the filter holder containing the capsules in an unused and sterile beaker, gently flush the capsules with 0.5 % (w/v) NaCl solution using a 25 mL graduated pipette (repeat 2 times)
+									</li>
+									<li>
+										Put the filter holder containing the capsules in an unused and sterile beaker, gently resuspend the capsules in the desired volume of storage medium
+									</li>
+								</ol>
-</div>
+							</div>
-</div>
+						</div>
+					</div>
+				</div>
+                <!-- Begin Entry -->
+				<div id="week15entry" class="nb-week" style="display: block">
+					<div class="twelve columns">
+						<div class="entry pc-media">
+							<div>
+        						<h3 class="centered">
+    							Preparation of samples
+        						</h3>
+								<ol>
+									<li>
+										Overnight inoculation at 37oC of each biological replicate of devices 1-3 + positive and negative controls in 3 ml LB + Kanamycin (Chloramphenicol for the controls)
+									</li>
+									<li>
+% inoculation at 37oC in 3 ml LB + Kanamycin (Chloramphenicol for the controls)
+									</li>
+									<li>
+										Measure OD, and dilute/adjust OD to 0.3 (+/- 0.025). This was found (by trial and error) to be a suitable density of cells for confocal microscopy.
+									</li>
+									<li>
+										Cell cultures were then put on ice and transported to the microscope.
+									</li>
+									<li>
+										For each biological replicate, 1.5 μl of the sample was put on a rectangular cover glass of thickness 1.5 (served as a suitable object glass for confocal microscopy). A small square-shaped cover glass of thickness 1.5 was put on top of the sample. To reduce bacteria moving and being pulled towards the edges of the cover glass, its edges were sealed with nail polish.
+									</li>
+									<li>
+										For each prepared microscopy sample (biological replicate), three images were obtained to serve as technical replicates. These three images should be taken at different xy positions in the prepared microscopy sample.
+									</li>
+								</ol>
+								<p>NOTE: The small volume of 1.5 μl ensures a short enough distance between the glasses (to work well with the z section size set by the pinhole size, see below), so that all bacteria between the glasses in a given xy(z) frame can be in focus and thus their fluorescence be detected by the microscope.</p>
+							</div>
+						</div>
+					</div>
+				</div>
+				<!-- End Entry -->
+                <!-- Begin Entry -->
+				<div id="week16entry" class="nb-week" style="display: block">
+					<div class="twelve columns">
+						<div class="entry pc-media">
+							<div>
+        						<h3 class="centered">
+    							Acquisition of images by confocal microscopy
+        						</h3>
+								<ol>
+									<li>
+										A Leica SP5 confocal microscope was used with the following parameters set in the LAS AF software: Argon laser (power 20 %) excitation line 488 nm (set to 6%), hybrid detector (HyD, Leica) set to filter and detect emitted light between 495-600 nm, pinhole size 399.99 nm (with a corresponding z section thickness of 2.897 μm).
+									</li>
+									<li>
+										The objective used was HCX PL APO CS (63x magnification, water immersion, numerical aperture 1.2, XY resolution 163 and Z resolution 290 at 488 nm).
+									</li>
+									<li>
+										Look into the microscope to focus in on your bacteria (bright field).
+									</li>
+									<li>
+										For live view, set the format to 512 * 512 and speed to 400 Hz, and press ‘Live’.
+									</li>
+									<li>
+										Adjust the focus with the z-position wheel, and when a good view of the bacteria and their fluorescence has been found in live view, stop the live view to scan the image. The following parameters were set in the LAS AF software to acquire the images: bit-depth of 12 (found under the configuration tab), acquisition mode xyz, zoom 1.25, speed 100 Hz, format 2048 * 2048 (pixel size 96.15 nm * 96.15 nm, image size 196.83 μm * 196.83 μm), line average 3.
+									</li>
+									<li>
+										The Leica SP5 should be set to acquire both a fluorescence image (with the HyD detector) and a regular bright field image (it is important to set both channels to ‘Visible’). The settings for the bright field channel were Smart Gain 305 V, Smart Offset -2.5 %. The setting for the fluorescence channel was Smart Gain 10.0 % (the HyD detector sets the offset by itself).
+									</li>
+									<li>
+										Each captured image (containing TIFF files of both bright field and fluorescence images) should be saved automatically under the same experiment (LIF file). This enables the microscopist to export the LIF file, which is then ready for the subsequent analysis.
+									</li>
+								</ol>
+								<p>NOTE: A bit-depth of 12 was chosen in this Interlab study to ensure that the range would accommodate both the samples with strong fluorescence and weak fluorescence. A lower bit-depth like 8 bit would cause the images with strong fluorescence to be oversaturated because the range would simply not be wide enough (data lost before analysis).</p>
+							</div>
+						</div>
+					</div>
+				</div>
+				<!-- End Entry -->
+                <!-- Begin Entry -->
+				<div id="week17entry" class="nb-week" style="display: block">
+					<div class="twelve columns">
+						<div class="entry pc-media">
+							<div>
+        						<h3 class="centered">
+    							Quantitative image analysis
+        						</h3>
+								<ol>
+									<li>
+										The data set was provided by the microscope software in LIF format. This file has about 1GB size. While ImageJ can read LIF format, it does not manage the memory very well, and cannot load the images simultaneously. The method used to circumvent this issue is to convert the LIF file into many TIFF files. Each TIFF file contained one fluorescence image and one bright field image. bfconvert utility was downloaded to convert the Leica LIF files into a set of TIFF files. The utility was downloaded from <a href="http://downloads.openmicroscopy.org/bio-formats/5.1.3/">http://downloads.openmicroscopy.org/bio-formats/5.1.3/</a>.
+									</li>
+									<li>
+										The images were converted with the command line: <pre>$ ./bfconvert 150818\ Interlab\ Hyd2.lif %n.tiff</pre>
+									</li>
+									<li>
+										The resulting files have spaces in their name. This created problems for ImageJ. The spaces were converted into underscores with the following command: <pre>$ for file in *.tiff; do mv "$file" `echo $file | tr ' ' '_'` ; done</pre>
+									</li>
+									<li>
+										The ImageJ analysis was scripted into a macro file. The commands of the macro file are as described in the following instructions.
+									</li>
+									<li>
+										The TIFF image was opened with ImageJ.
+									</li>
+									<li>
+										The two stacks of the images were converted into two images for easy reference in the macro.
+									</li>
+									<li>
+										The bright field image was selected and viewed with a Grays LUT.
+									</li>
+									<li>
+										The background was subtracted using Process > Subtract background...". In the new window, the rolling ball radius is 50 px, and the "Light" was ticked. A new window appeared asking whether to process the images. "No" was clicked.
+									</li>
+									<li>
+										The threshold was determined by clicking Image > Adjust > Threshold… "Apply" was clicked. On the new window, Background: Light was selected. Calculate threshold for each image was unticked. "Apply" was clicked/.
+									</li>
+									<li>
+										Analyze > Set Measurements was selected, "Area", and "Mean" are ticked, and Redirect to: was set the name of the first image (fluorescence).
+									</li>
+									<li>
+										Analyze > Analyze Particles was clicked. For size, 2E-8 was set as the lower bound. For circularity, 0.10 to 0.7 was set, and show outline was selected for visualization purposes.
+									</li>
+									<li>
+										Results window was selected, and saved as a CSV file.
+									</li>
+									<li>
+										In MATLAB, all the batch processed files were converted into a 3D matrix TABLE (Devices, Biological replicates, Technical replicates), the intensity was calculated as <code>MEAN(TABLE,3)</code>, which is the average over the technical replicates. These results were satisfactorily compared with the flow cytometry results.
+									</li>
+								</ol>
+							</div>
+						</div>
+					</div>
+				</div>
+				<!-- End Entry -->
+                <!-- Begin Entry -->
+				<div id="week18entry" class="nb-week" style="display: block">
+					<div class="twelve columns">
+						<div class="entry pc-plas">
+							<div>
+        						<h3 class="centered">
+    							LB media
+        						</h3>
+								<ul>
+									<li>
+% Tryptone
+									</li>
+									<li>
+% Yeast extract
+									</li>
+									<li>
+% NaCl
+									</li>
+								</ul>
+								<ol>
+									<li>
+										Autoclave for 20 minutes at 120°C
+									</li>
+									<li>
+										Add appropriate antibiotics up to respective working concentration
+									</li>
+								</ol>
+							</div>
+						</div>
+					</div>
+				</div>
+				<!-- End Entry -->
+                <!-- Begin Entry -->
+				<div id="week19entry" class="nb-week" style="display: block">
+					<div class="twelve columns">
+						<div class="entry pc-plas">
+							<div>
+        						<h3 class="centered">
+    							LA media
+        						</h3>
+								<ul>
-<div id="week6entry" class="nb-week" style="display: block">
+									<li>
-				<div class="twelve columns">
+% Tryptone
-					<h3 class="centered">Gibson Assembly</h3>
+									</li>
-<div class="entry pc-techniques">
+									<li>
-	<div>
+% Yeast extract
-		<h6>
+									</li>
-					</h6>
+									<li>
-		<div class="nb-tech">{{{tech}}}</div>
+% NaCl
-			<p>
+									</li>
-			The Gibson assembly protocol can be found at <a href="https://www.neb.com/protocols/2012/12/11/gibson-assembly-protocol-e5510">New England Biolabs</a>.
+									<li>
-			</p>
+% Agar
-		</div>
+									</li>
-	</div>
+									<li>
-</div>
+										Autoclave for 20 minutes at 120°C
-</div>
+									</li>
+									<li>
+										Add appropriate antibiotics up to respective working concentration
+									</li>
+								</ul>
+							</div>
+						</div>
+					</div>
+				</div>
+				<!-- End Entry -->
+                <!-- Begin Entry -->
+				<div id="week20entry" class="nb-week" style="display: block">
+					<div class="twelve columns">
+						<div class="entry pc-plas">
+							<div>
+        						<h3 class="centered">
+    							Antibiotic stocks (according to iGEM protocol)
+        						</h3>
+								<ul>
-<div id="week7entry" class="nb-week" style="display: block">
+									<li>
-				<div class="twelve columns">
+										Ampicillin: 100mg/ml in 50% EtOH
-					<h3 class="centered">DNA isolation and cleaning</h3>
+									</li>
-</p>
+									<li>
-<div class="entry pc-techniques">
+										Chloramphenicol: 35mg/ml in 100% EtOH
-	<div>
+									</li>
+									<li>
+										Kanamycin: 35mg/ml in distilled H20
+									</li>
+									<li>
+										Aliquots of 500ul, stored at -20°C
+									</li>
+								</ul>							</div>
+						</div>
+					</div>
+				</div>
+				<!-- End Entry -->
+                <!-- Begin Entry -->
-		<div class="nb-tech">{{{tech}}}</div>
+				<div id="week21entry" class="nb-week" style="display: block">
-<p>		For plasmid isolation, the Promega Wizard Plus SV Minipreps DNA Purification System A1460 Miniprep protocol was used. For PCR product isolation, the  QIAquick PCR Purification kit was used.
+					<div class="twelve columns">
-</p>
+						<div class="entry pc-plas">
-	</div>
+							<div>
-</div>
+        						<h3 class="centered">
+    							Making P. putida electrocompetent
+        						</h3>
+								<ol>
-</div>
+									<li>
-</div>
+										Inoculate of 1% from ON culture in 10ml LB media
+									</li>
+									<li>
+										Incubate at 30°C until OD600=0.45+-10%
+									</li>
+									<li>
+										Aliquot 1.5ml cell suspension in an eppendorf tube per electroporation
+									</li>
+									<li>
+										Centrifugate at 12000g for 1 minute at 4°C
+									</li>
+									<li>
+										Remove supernatant
+									</li>
+									<li>
+										Add 1ml 300mM sucrose (cold)
+									</li>
+									<li>
+										Centrifugate at 12000g for 1 minute at 4°C
+									</li>
+									<li>
+										Remove supernatant
+									</li>
+									<li>
+										Add 50ul 300mM sucrose (cold)
+									</li>
+									<li>
+										Keep on ice until used for electroporation
+									</li>
+								</ol>
-<div id="week8entry" class="nb-week" style="display: block">
+							</div>
-				<div class="twelve columns">
+						</div>
-					<h3 class="centered">DNA digestion</h3>
+					</div>
-</p>
+				</div>
-<div class="entry pc-techniques">
+				<!-- End Entry -->
-	<div>
+                <!-- Begin Entry -->
-		<div class="nb-tech">{{{tech}}}</div>
+				<div id="week22entry" class="nb-week" style="display: block">
-<p>
+					<div class="twelve columns">
+						<div class="entry pc-plas">
+							<div>
+        						<h3 class="centered">
+    							Electroporation
+        						</h3>
-DNA digests for both ligation and verification used the protocol in the <a href="http://unitedscientists.org/labs/norway/NTNU/PhotoSynLab/wiki/table-of-contents-2/protocol-molecular/dna-ligation/restriction-enzyme-digest">PhotoSynLab wiki</a>.
+								<ol>
+									<li>
+										Add plasmid (1-5ul, up to 500ng DNA) to 50ul electrocompetent cells into electroporation
+									</li>
+									<li>
+										Electroporation with Biorad GenePulser Xcell
+									</li>
+									<ul>
+										<li>
+											Voltage: 2500V
+										</li>
+										<li>
+											Capacitance: 25uF
+										</li>
+										<li>
+											Resistence: 200(Ohm)
+										</li>
+										<li>
+											    Cuvette: 2mm)
+										</li>
+									</ul>
+									<li>
+										Add 1ml LB media to the electroporation chamber immediately after pulse
+									</li>
+									<li>
+										Transfer all liquid from the electroporation chamber into an eppendorf tube
+									</li>
+									<li>
+										Incubate for 1 hour at 30°C
+									</li>
+									<li>
+										Plate cells
+									</li>
+								</ol>
+							</div>
+						</div>
+					</div>
+				</div>
+				<!-- End Entry -->
-	</div>
+                <!-- Begin Entry -->
-</div>
+				<div id="week23entry" class="nb-week" style="display: block">
+					<div class="twelve columns">
+						<div class="entry pc-plas">
+							<div>
+        						<h3 class="centered">
+    							Plating
+        						</h3>
+								<ol>
-</div>
+									<li>
-</div>
+										Transfer 50ul of cell suspension in the middle of the LA plate (with appropriate antibiotic)
+									</li>
+									<li>
+										Distribute cells equally over the whole plate
+									</li>
+									<li>
+										Centrifuge remaining cell suspension and remove supernatant until less then 100ul remains
+									</li>
+									<li>
+										Resuspend cells in remaining supernatant
+									</li>
+									<li>
+										Transfer all remaining cell suspension onto a second LA plate
+									</li>
+									<li>
+										Distribute cells equally over the whole plate
+									</li>
+								</ol>
+							</div>
+						</div>
+					</div>
+				</div>
+				<!-- End Entry -->
+                <!-- Begin Entry -->
+				<div id="week24entry" class="nb-week" style="display: block">
+					<div class="twelve columns">
+						<div class="entry pc-plas">
+							<div>
+        						<h3 class="centered">
+    							Heat transformation of E.coli
+        						</h3>
+								<ol>
-<div id="week9entry" class="nb-week" style="display: block">
+									<li>
-				<div class="twelve columns">
+										Add plasmid (1-5ul, up to 500ng DNA) to 100ul competent cells (stored at -80°C) in eppendorf tube
-					<h3 class="centered">PCR</h3>
+									</li>
-</p>
+									<li>
-<div class="entry pc-techniques">
+										Keep on ice for 30 minutes
-	<div>
+									</li>
+									<li>
-		<div class="nb-tech">{{{tech}}}</div>
+										Keep in water bath for 45 seconds at 42°C
-<p>		The touchdown PCR procedure detailed on the <a href="http://unitedscientists.org/labs/norway/NTNU/PhotoSynLab/wiki/table-of-contents-2/protocol-molecular/pcr-touchdown-pcr">PhotoSynLab wiki</a> was used to amplify DNA.
+									</li>
-</p>
+									<li>
-	</div>
+										Keep on ice for 3 minutes
-</div>
+									</li>
+									<li>
+										Add 500ul LB media
+									</li>
+									<li>
+										Incubate for 1 hour at 37°C
+									</li>
+								</ol>
+							</div>
+						</div>
+					</div>
+				</div>
+				<!-- End Entry -->
-</div>
+                <!-- Begin Entry -->
-</div>
+				<div id="week25entry" class="nb-week" style="display: block">
+					<div class="twelve columns">
+						<div class="entry pc-plas">
+							<div>
+        						<h3 class="centered">
+    							Miniprep: Preparation of DNA from E.coli
+        						</h3>
+								<p>According to Wizard Plus SV Minipreps DNA Purification system</p>
+							</div>
+						</div>
+					</div>
+				</div>
+				<!-- End Entry -->
-<div id="week10entry" class="nb-week" style="display: block">
+                <!-- Begin Entry -->
-				<div class="twelve columns">
+				<div id="week26entry" class="nb-week" style="display: block">
-					<h3 class="centered">Nanodrop</h3>
+					<div class="twelve columns">
-</p>
+						<div class="entry pc-plas">
-<div class="entry pc-techniques">
+							<div>
-	<div>
+        						<h3 class="centered">
+    							Nanodrop: Measurement of DNA concentration
-		<div class="nb-tech">{{{tech}}}</div>
+        						</h3>
-<p>		<a href="http://www.nanodrop.com/library/nd-1000-v3.7-users-manual-8.5x11.pdf">A NanoDrop ND-1000 Spectrophotometer</a> was used to determine DNA concentrations.
-</p>
-	</div>
-</div>
-</div>
-</div>
-<div id="week11entry" class="nb-week" style="display: block">
+							</div>
-				<div class="twelve columns">
+						</div>
-					<h3 class="centered">3A assembly</h3>
+					</div>
-</p>
+				</div>
-<div class="entry pc-techniques">
+				<!-- End Entry -->
-	<div>
-		<div class="nb-tech">{{{tech}}}</div>
-<p>		3A assembly was conducted according to the  <a href="http://parts.igem.org/Help:Protocol/3A_Assembly">iGEM 3A assembly protocol</a>.
-</p>
-	</div>
-</div>
-</div>
+                <!-- Begin Entry -->
-</div>
+				<div id="week27entry" class="nb-week" style="display: block">
+					<div class="twelve columns">
+						<div class="entry pc-plas">
+							<div>
+        						<h3 class="centered">
+    							Enzyme digest (according to iGEM protocol)
+        						</h3>
+								<ol>
+									<li>
+										Keep all enzymes and buffers used on ice
+									</li>
+									<li>
+										Add 250ng of DNA to 3ul of Cutsmart Buffer and 0.5ul of each appropriate enzyme. Fill up with nuclease free water to a total volume of 16ul
+									</li>
+									<li>
+										Incubate mixture for 30 minutes at 37°C (enzyme digest)
+									</li>
+									<li>
+										Incubate mixture for 20 minutes at 80°C (heat inactivation)
+									</li>
+								</ol>
-<div id="week12entry" class="nb-week" style="display: block">
+							</div>
-				<div class="twelve columns">
+						</div>
-					<h3 class="centered">Ligation</h3>
+					</div>
+				</div>
+				<!-- End Entry -->
-<div class="entry pc-techniques">
+                <!-- Begin Entry -->
-	<div>
+				<div id="week28entry" class="nb-week" style="display: block">
+					<div class="twelve columns">
-		<div class="nb-tech">{{{tech}}}</div>
+						<div class="entry pc-plas">
-<p>		Ligation was performed according to the protocol outlined on the  <a href="http://unitedscientists.org/labs/norway/NTNU/PhotoSynLab/wiki/table-of-contents-2/protocol-molecular/pcr-touchdown-pcr">PhotoSynLab wiki</a>.
+							<div>
-</p>
+        						<h3 class="centered">
-	</div>
+    							Ligation (according to iGEM protocol)
-</div>
+        						</h3>
+								<ol>
-</div>
+									<li>
-</div>
+										Add 25ng of DNA to 1ul T4 DNA Ligase buffer and 0.5ul T4 DNA Ligase. Fill up with nuclease free water to a total colume of 10ul
+									</li>
+									<li>
+										Incubate for 30 minutes at 16°C (ligation)		*or overnight
+									</li>
+									<li>
+										Incubate for 20 minutes at 65°C (heat inactivation)
+									</li>
+									<li>
+										Transformation with 2ul of product
+									</li>
+								</ol>
+							</div>
+						</div>
+					</div>
+				</div>
+				<!-- End Entry -->
+                <!-- Begin Entry -->
+				<div id="week29entry" class="nb-week" style="display: block">
+					<div class="twelve columns">
+						<div class="entry pc-plas">
+							<div>
+        						<h3 class="centered">
+    							Polymerase Chain Reaction (PCR)
+        						</h3>
+								<h5>Recipe</h5>
+								<ul>
-<div id="week13entry" class="nb-week" style="display: block">
+									<li>
-				<div class="twelve columns">
+.5ul forward primer
-					<h3 class="centered">Transformation (Escherichia coli)</h3>
+									</li>
-</p>
+									<li>
-<div class="entry pc-techniques">
+.5ul reverse primer
-	<div>
+									</li>
+									<li>
-		<div class="nb-tech">{{{tech}}}</div>
+ul dNTP
-<p>		The protocols used for preparing competent DH5a cells, as well as the heat-shock transformation procedure employed can be found at the <a href="http://unitedscientists.org/labs/norway/NTNU/PhotoSynLab/wiki/table-of-contents-2/protocol-molecular/heat-shock-transformation-2">PhotoSynLab wiki</a>.
+									</li>
-</p>
+									<li>
-	</div>
+										<1000ng template DNA
-</div>
+									</li>
+									<li>
+.5ul Q5 polymerase
+									</li>
+									<li>
+ul Q5 buffer
+									</li>
+									<li>
+ul High GC enhancer
+									</li>
+									<li>
+										Fill up with nuclease free water to a total volume of 50ul
+									</li>
+								</ul>
+								<h5>Protocol</h5>
+								<ol>
-</div>
+									<li>
-</div>
+seconds - 98°C
+									</li>
+									<li>
+seconds - 98°C
+									</li>
+									<li>
+seconds - Annealing temperature
+									</li>
+									<li>
+seconds - 72°C
+									</li>
+									<li>
+										Repeat steps 2.-4. 20-30 times
+									</li>
+									<li>
+minutes - 72°C
+									</li>
+									<li>
+										Infinite hold at 4°C
+									</li>
+								</ol>
-<div id="week14entry" class="nb-week" style="display: block">
+							</div>
-				<div class="twelve columns">
+						</div>
-					<h3 class="centered">Transformation (Synechocystis sp. PCC 6803)</h3>
+					</div>
-</p>
+				</div>
-<div class="entry pc-techniques">
+				<!-- End Entry -->
-	<div>
-		<div class="nb-tech">{{{tech}}}</div>
-<p>		The transformation procedure for transforming Synechocystis can be found at the <a href="http://unitedscientists.org/labs/norway/NTNU/PhotoSynLab/wiki/table-of-contents-2/protocol-molecular/synechocystis-transformation-r-hill-method">PhotoSynLab wiki</a>.
-</p>
-	</div>
-</div>
-</div>
+                <!-- Begin Entry -->
-</div>
+				<div id="week30entry" class="nb-week" style="display: block">
+					<div class="twelve columns">
+						<div class="entry pc-plas">
+							<div>
+        						<h3 class="centered">
+    							Gelelectrophoresis
+        						</h3>
-<div id="week16entry" class="nb-week" style="display: block">
-				<div class="twelve columns">
-					<h3 class="centered">Right flank</h3>
-<div class="entry pc-plas">
+							</div>
-	<div>
+						</div>
+					</div>
-		<div class="nb-tech">{{{tech}}}</div>
+				</div>
-<p>	<a href="http://parts.igem.org/Part:BBa_K1424001">BBa_K1424001</a>.
+				<!-- End Entry -->
-</p>
-	</div>
-</div>
-</div>
+                <!-- Begin Entry -->
-</div>
+				<div id="week31entry" class="nb-week" style="display: block">
+					<div class="twelve columns">
+						<div class="entry pc-plas">
+							<div>
+        						<h3 class="centered">
+    							Spectrophotometer
+        						</h3>
-<div id="week17entry" class="nb-week" style="display: block">
-				<div class="twelve columns">
-					<h3 class="centered">Left flank</h3>
-<div class="entry pc-plas">
+							</div>
-	<div>
+						</div>
+					</div>
-		<div class="nb-tech">{{{tech}}}</div>
+				</div>
-<p>	<a href="http://parts.igem.org/Part:BBa_K1424000">BBa_K1424000</a>.
+				<!-- End Entry -->
-</p>
-	</div>
-</div>
-</div>
+                <!-- Begin Entry -->
-</div>
+				<div id="week32entry" class="nb-week" style="display: block">
+					<div class="twelve columns">
+						<div class="entry pc-plas">
+							<div>
+        						<h3 class="centered">
+    							Gel extraction
+        						</h3>
-<div id="week18entry" class="nb-week" style="display: block">
-				<div class="twelve columns">
-					<h3 class="centered">Kanamycin resistance</h3>
-<div class="entry pc-plas">
+							</div>
-	<div>
+						</div>
+					</div>
-		<div class="nb-tech">{{{tech}}}</div>
+				</div>
-<p>	<a href="http://parts.igem.org/Part:BBa_K1424003">BBa_K1424003</a>.
+				<!-- End Entry -->
-</p>
-	</div>
-</div>
-</div>
+                <!-- Begin Entry -->
-</div>
+				<div id="week33entry" class="nb-week" style="display: block">
+					<div class="twelve columns">
+						<div class="entry pc-plas">
+							<div>
+        						<h3 class="centered">
+    							Flow Cytometry
+        						</h3>
+								<ol>
-<div id="week19entry" class="nb-week" style="display: block">
+									<li>
-				<div class="twelve columns">
+										Inoculate 1% of overnight culture in 3ml LB media
-					<h3 class="centered">Lac promotor</h3>
+									</li>
+									<li>
+										Incubate at the appropriate temperature (30°C for P.putida, 37°C for E.coli)
+									</li>
+									<li>
+										Dilute 1:100 in PBS, total volume: 1.5ml
+									</li>
+									<li>
+										Transfer 500ul in flow cytometry tube (per technical replicate)
+									</li>
+									<li>
+										Flow cytometry with BD Accuri C6 sampler
+									</li>
+									<ul>
+										<li>
+											Suction: 2 minutes
+										</li>
+										<li>
+											Flow rate: 35ul/minute
+										</li>
+										<li>
+											Pre-set thresholds: FSC=4000, FL-1=200
+										</li>
+										<li>
+											Washing steps after last technical replicate from each sample
+										</li>
+									</ul>
+								</ol>
-<div class="entry pc-plas">
-	<div>
-		<div class="nb-tech">{{{tech}}}</div>
-<p>	<a href="http://parts.igem.org/Part:BBa_K1424002">BBa_K1424002</a>.
-</p>
-	</div>
-</div>
-</div>
+							</div>
-</div>
+						</div>
+					</div>
+				</div>
+				<!-- End Entry -->
-<div id="week20entry" class="nb-week" style="display: block">
+                <!-- Begin Entry -->
-				<div class="twelve columns">
+				<div id="week34entry" class="nb-week" style="display: block">
-					<h3 class="centered">Glucose oxidase</h3>
+					<div class="twelve columns">
+						<div class="entry pc-plas">
+							<div>
+        						<h3 class="centered">
+    							PBS
+        						</h3>
-<div class="entry pc-plas">
-	<div>
-		<div class="nb-tech">{{{tech}}}</div>
-<p>	<a href="http://parts.igem.org/Part:BBa_K1424004">BBa_K1424004</a>.
-</p>
-	</div>
-</div>
-</div>
+							</div>
-</div>
+						</div>
+					</div>
+				</div>
+				<!-- End Entry -->
-<div id="week21entry" class="nb-week" style="display: block">
+                <!-- Begin Entry -->
-				<div class="twelve columns">
+				<div id="week35entry" class="nb-week" style="display: block">
-					<h3 class="centered">Left flank + Kanamycin resistance + Right flank</h3>
+					<div class="twelve columns">
+						<div class="entry pc-plas">
+							<div>
+        						<h3 class="centered">
+    							HIFI
+        						</h3>
-<div class="entry pc-plas">
-	<div>
-		<div class="nb-tech">{{{tech}}}</div>
-<p>	<a href="http://parts.igem.org/Part:BBa_K1424005">BBa_K1424005</a>.
-</p>
-	</div>
-</div>
-</div>
+							</div>
-</div>
+						</div>
+					</div>
+				</div>
+				<!-- End Entry -->
+                <!-- Begin Entry -->
+				<div id="week36entry" class="nb-week" style="display: block">
+					<div class="twelve columns">
+						<div class="entry pc-plas">
+							<div>
+        						<h3 class="centered">
+    							TE Buffer
+        						</h3>
+							</div>
+						</div>
+					</div>
+				</div>
+				<!-- End Entry -->
+			</div>
-<div id="week24entry" class="nb-week" style="display: block">
-				<div class="twelve columns">
-					<h3 class="centered">DNA concentration</h3>
-<div class="entry pc-calc">
-	<div>
-		<div class="nb-tech">{{{tech}}}</div>
-<p> After measuring the concentration of a sample of DNA in ng/ul with the NanoDrop method, the concentration in terms of pmolar could be determined with the equation
-<img src="https://static.igem.org/mediawiki/2014/c/c5/Eqn3.gif">
-</p>
-	</div>
-</div>
 </div>