Research work
Finding a suitable topic was very challenging and time consuming. Initially, we looked through projects of prior teams and assembled a list of possible topics.
A big influence was a new method for assembling genes in a manufacturing manner which was being developed by a research group on our university. Based on the quick and easy synthesis of proteins a first idea was the creation of enzymes that could convert blood groups. Also working with cyanobacteria was an option we considered.
After many seminars, we established the idea of metabolic channeling using dCas9 as our main project. One of our advisors also worked with membraneless organelles and suggested this approach for achieving metabolic channeling and therefor our secondary project with LLPS.
We thought about using either violacein or beta carotene as exemplary pathways for our increased production but finally decided for beta carotene. This brought many new challenges in the form of understanding the pathway and implementing it in E. coli.
Also, we very worried that an increased output would end up consuming too much precursor substrate and hinder growth of the transformed cells. Additionally, we found that team Edinburgh/Glasgow had problems with toxicity if the enzymes of the beta carotene pathway were in a specific order.
But all the planning was for nothing when we realized that some of the enzymes of the beta carotene pathway were localized in the membrane and therefore not suitable for our metabolic channeling approach.
After planning the design more precise we eventually arrived at our scaffold design of a low and a high-copy plasmid.
2. This preparation step is needed to create sticky ends on the cassettes.
3. This step is only performed once.
4. Restriction with EcoRI and PstI (see restriction protocol) for all components.
plasmid | cassettes | ||
---|---|---|---|
psB1C3 | 1 | 2 | 3 |
psB1A3 | 1 | 2 | 3 |
psB1K3 | 1 | 2 | 3 |
2. Selection with corresponding antibiotics.
2. Miniprep and check concentration via nanodrop.
3. Many aliquots needed (small volume because thawing time) for future reactions!
2. 3A-assembly
2. It is important to only combine cassettes with the same number (1, 2 and 3 have varying spacer length).
3. We will add cassettes and test frequently for viability to determine the maximum target-sequence length.
4. We want to combine about five cassettes.
2. In the first step, we can either just use the prepared plasmid with cassettes already inserted or use an empty one, because the part in between will be cut out anyway.
3. The be able to select for plasmid with higher cassette content the resistances will cycle.
4. The resistance cycle (for plasmids) is K C A.
5. For the inserts, the resistance signals from which plasmid they will be cut.
6. The plasmids resistance determines the selection antibiotics for that step!
AX – Ampicillin (Insert/Plasmid) from step X
KX – Kanamycin (Insert/Plasmid) from step X
E – EcoRI
S – SpeI
X – XbaI
P – PstI
Insert 1 E+S | Insert 2 X+P | Plasmid E+P |
---|---|---|
C1 | A1 | K1 |
A2 | K1 | C2 |
K3 | C2 | A3 |
C4 | A3 | K4 |
2. Insert 1 and 2 are ligated into plasmid K1.
3. Insert 1 is cut out of plasmid A2/Insert 2 (here the fusion from Insert 1+2 from the first cycle) is cut out of K1 and plasmid C2 is linearized.
4. Insert 1 and 2 are ligated into plasmid C2.
5. With insert 1 coming from K3 and insert 2 from C2 (fusion) the steps will repeated until maximum number of inserts is reached.
6. The assembly needs to be done for all 3 cassettes simultaneously after each step.
7. Transformation of ligation into competent cells (see transformation protocol).
8. Selection with corresponding antibiotics.
2. Miniprep and check concentration via nanodrop.
[1] http://parts.igem.org/Help:Protocols/3A_Assembly (accessed 31 October 2017)
2. Good to know before the start
2. Include a no-template control and positive control in parallel.
3. Thaw and keep reagents on ice.
4. Mix well before use.
5. The longer the amplicon, the longer the extension time: Use 15 sec/kb extension.
6. Use 90 sec extension for multiplexing.
7. Run an annealing temperature gradient from 55 °C to 65 °C to choose the best specificity conditions. Do not use fast cycling for multiplexing.
8. ALLin™ Red Taq Mastermix, 2X is premixed with red dye and density reagents for direct loading on the gels after the PCR. In a 2 % agarose TAE gel the dye migrates with ~350 bp DNA, in 1 % agarose TAE gel with ~600 bp DNA fragments.
3. Are you working with A. E.coli or B. yeast?
Steps | Cycles | Temperature | Time |
---|---|---|---|
Initial denaturation |
1 cycle |
95°C | 60s |
Denaturation | 30-40 cycles |
95°C | 15s |
Annealing | 30-40 cycles |
55-65°C | 15s |
Extension | 30-40 cycles |
72°C | 15-90s (15 sec per 1 kb) |
Final extension | 1 cycle |
72°C | 5 min |
1. If resuspended colonies are to be used: pipette 50 μl of a 0.02 M NaOH solution into each of a set of appropriately labelled PCR tubes or wells of a PCR plate. Using sterile pipette tips or toothpicks, transfer transformants to individual tubes/wells. The amount of cells resuspended must just be visible. Resuspend cells by pipetting or vortexing and incubate for ≥ 5 min at 37 °C.
2. If overnight cultures are to be used: pipette 40 μl of a 0.01 M NaOH solution into each of a set of appropriately labelled PCR tubes or wells of a PCR plate. Transfer 10 μl of each overnight culture to be tested to the appropriate tube/well and mix by pipetting up and down. Incubate for ≥ 5 min at 37 °C.
3. Prepare a PCR master mix (always prepare at least 10 % more).
4. Aliquot 22.5 μl of PCR master mix into each PCR tube.
5. Add 2.5 μl of the resuspended colony or overnight culture mixed with NaOH to the appropriate PCR tube.
6. Close the tubes
7. Perform the PCR using the following cycling profile:
Steps | Cycles | Temperature | Time |
---|---|---|---|
Initial denaturation |
1 cycle |
95°C | 60s |
Denaturation | 30-40 cycles |
95°C | 15s |
Annealing | 30-40 cycles |
55-65°C | 15s |
Extension | 30-40 cycles |
72°C | 15-90s |
Final extension | 1 cycle |
72°C | 5 min |
[1]https://www.highqu.com/media/wysiwyg/ressources/manuals/PCM02_ALLin_Red_Taq_Mastermix_PI.pdf (accessed 31 October 2017)
1. Aim
2. Steps
1. Depending on the PCR product
If there is more than the wanted DNA band: A. Dissolving the Gel Slice |
If there iss only one DNA band: B. Processing PCR Amplifications |
---|---|
1. Following electrophoresis, excise DNA band from gel (with scalpel) and place gel slice in a 1.5 ml microcentrifuge tube. 2. Add 10 μl Membrane Binding Solution per 10 mg of gel slice. Vortex and incubate at 50 – 65 °C until gel slice is completely dissolved. |
You can just work with the rest of your PCR aliquote (when you did not use all of it for the gel electrophoresis). |
2. Transfer dissolved gel mixture or prepared PCR product to the Minicolumn assembly. Incubate at room temperature for 1 minute.
3. Centrifuge at 16,000 ×g for 1 minute. Discard flowthrough and reinsert Minicolumn into Collection Tube.
4. Heat NE-buffer to 70 °C.
3. Washing
2. Repeat Step 4 with 500 μl Membrane Wash Solution. Centrifuge at 16,000 × g for 5 minutes.
3. Empty the Collection Tube and recentrifuge the column assembly for 1 minute with the microcentrifuge lid open(or off to allow evaporation of any residual ethanol.
4. Elution
2. Add 50 μl of Nuclease-Free Water to the Minicolumn. Incubate at room temperature for 1 minute. Centrifuge at 16,000 × g for 1 minute.
3. Discard Minicolumn and measure the concentration.
4. Store DNA at 4°C or –20°C.
[1]https://www.promega.de/-/media/files/resources/protcards/wizard-sv-gel-and-pcr-clean-up-system-quick-protocol.pdf?la=de-de (accessed 31 October 2017)
1. What is it ?
2. Uses an electrical field to move the negatively charged DNA through an agarose gel matrix toward a positive electrode.
3. Shorter DNA fragments migrate through the gel more quickly than longer ones.
2. Why are we doing it ?
Note: Agarose gels are commonly used in concentrations of 0.7 % to 2 % depending on the size of bands needed to be separated - Simply adjust the mass of agarose in a given volume to make gels of other agarose concentrations (e.g., 2 g of agarose in 100 mL of TAE will make a 2 % gel).
2. Microwave for 1-3 min until the agarose is completely dissolved (but do not overboil the solution, as some of the buffer will evaporate and thus alter the final percentage of agarose in the gel. Many people prefer to microwave in pulses, swirling the flask occasionally as the solution heats up.).
Note: gloves and glasses ! Caution HOT! Be careful stirring, eruptive boiling can occur.
It is a good idea to microwave for 30-45 sec, stop and swirl, and then continue towards a boil. Keep an eye on it as the initial boil has a tendency to boil over. Placing saran wrap over the top of the flask can help with this, but is not necessary if you pay close attention.
4. Pouring of the gel
Note: Or cool down in water bath about 30 min.
2. Add ethidium bromide (EtBr) to a final concentration of approximately 0.2-0.5 μg/mL (usually about 2-3 μl of lab stock solution per 100 mL gel). EtBr binds to the DNA and allows you to visualize the DNA under ultraviolet (UV) light.
Note: Caution EtBr is a known mutagen. Wear a lab coat, eye protection and gloves when working with this chemical. If you add EtBr to your gel, you will also want to add it to the running buffer when you run the gel.
3. Pour the agarose into a gel tray with the well comb in place.
Note: Think about witch gel tray size you need (a small one or a big one).
Pour slowly to avoid bubbles which will disrupt the gel. Any bubbles can be pushed away from the well comb or towards the sides/edges of the gel with a pipette trip.
4. Let the newly poured gel sit at room temperature for 20-30 mins, until it has completely solidified.
if you are in a hurry the gel can also be set more quickly if you place the gel tray at 4 °C earlier so that it is already cold when the gel is poured into it.
5. Loading Samples and Running an Agarose Gel
Note: Loading buffer serves two purposes: 1) it provides a visible dye that helps with gel loading and will also allows you to gauge how far the gel has run while you are running your gel; and 2) it contains a high percentage of glycerol, so it increases the density of your DNA sample causing it settle to the bottom of the gel well, instead of diffusing in the buffer.
2. Once solidified, place the agarose gel into the gel box (electrophoresis unit).
3. Fill gel box with 1xTAE (or TBE) until the gel is covered.
4. Carefully load a molecular weight ladder into the first lane of the gel.
Note: When loading the sample in the well, maintain positive pressure on the sample to prevent bubbles or buffer from entering the tip. Place the very top of the tip of the pipette into the buffer just above the well. Very slowly and steadily, push the sample out and watch as the sample fills the well. After all of the sample is unloaded, push the pipettor to the second stop and carefully raising the pipette straight out of the buffer.
5. Carefully load your samples into the additional wells of the gel.
6. Run the gel at 80-150 V until the dye line is approximately 75-80 % of the way down the gel.
Note: Black is negative, red is positive. (The DNA is negatively charged and will run towards the positive electrode.) Always Run to Red.
Note: A typical run time is about 1-1.5 hours, depending on the gel concentration and voltage.
7. Turn OFF power, disconnect the electrodes from the power source, and then carefully remove the gel from the gel box.
8. Using any device that has UV light, visualize your DNA fragments.
Note: When using UV light, protect your skin by wearing safety goggles or a face shield, gloves and a lab coat.
Note: If you will be purifying the DNA for later use, use long-wavelength UV and expose for as little time as possible to minimize damage to the DNA.
Note: The fragments of DNA are usually referred to as ‘bands’ due to their appearance on the gel.
6. Analyzing Your Gel
7. Purifying DNA from Your Gel
[1] http://www.addgene.org/protocols/gel-electrophoresis/ (accessed 31 October 2017)
1. Aim
component | volume |
---|---|
T4 DNA Ligase Buffer (10 x) | 2 µl |
10x buffer | 1 µl |
T4 DNA Ligase | 1 µl |
Vector DNA | |
Insert DNA | |
Nuclease-free water | to 20 µl |
kb (smaller DNA)/ kb (larger DNA)⋅mass (Vector DNA)⋅relation (Insert DNA)
Example calculation
mass Vector DNA: 100 ng
Vector DNA: 10 kb
Insert DNA: 3 kb
3 kb/ 10 kb⋅100 ng⋅3 = 90 ng
3. Use nebiocalculator.neb.com/#!/ to calculate molar ratios.
4. The T4 DNA Ligase Buffer should be thawed and resuspended at room temperature.
3. Incubation
5. Chill on ice and transform 1-5 μl of the reaction into 50 μl competent cells.
[1] https://www.neb.com/protocols/1/01/01/dna-ligation-with-t4-dna-ligase-m0202 (accessed 31 October 2017)
1. Aim
2.Production of cleared lysate
2. Centrifugation for 5 minutes at 10,000 xg in a tabletop centrifuge.
3. Pour off the supernatant.
4. Reinsert again bacterial culture to the pellet and repeat step 2 and 3.
5. Blot the inverted tube on a paper towel to remove excess media.
2. Completely resuspend the cell pellet by vortexing or pipetting.
3. It is essential to thoroughly resuspend the cells.
2. Mix by inverting the tube 4 times - do not vortex.
3. Incubate until the cell suspension clears (clear ≠ colorlessly) (approximately 1–5 minutes).
2. Mix by inverting the tube 4 times - do not vortex.
3. Incubate for 5 minutes at room temperature.
2. Immediately mix by inverting the tube 4 times - do not vortex.
2. Centrifuge at maximum speed in a microcentrifuge for 1 minute at room temperature.
3. Remove the Spin Column from the tube and discard the flowthrough.
4. Reinsert the Spin Column into the Collection Tube.
2. Centrifuge at maximum speed in a microcentrifuge for 2 minutes at room temperature.
3. If the Spin Column has Column Wash Solution associated with it, centrifuge again for 1 minute at maximum speed.
4. Transfer the Spin Column to a new, sterile 1.5ml microcentrifuge tube, being careful not to transfer any of the Column Wash Solution with the Spin Column.
2. Centrifuge at maximum speed for 1 minute at room temperature in a microcentrifuge.
[1]https://www.promega.de/-/media/files/resources/protocols/technical-bulletins/0/wizard-plus-sv-minipreps-dna-purification-system-protocol.pdf (accessed 31 October 2017)
1. What is the PCR ?
polymerase chain reaction
Method to make multiple copies of a the specific DNA-sequence
2. Reaction Setup
2. Preheat the thermocycler to the denaturation temperature (98 °C).
3. Prior to use all components should be mixed.
4. Work quickly when transferring the reactions to a thermocycler.
3. Steps
Component | 25 μl Reaction | 50 μl Reaction | Final Concentration |
---|---|---|---|
Q5 High-Fidelity 2X Master Mix | 12.5 μl | 25 μl | 1X |
10 μM Forward Primer | 1.25 μl | 2.5 μl | 0.5 μM |
10 μM Reverse Primer | 1.25 μl | 2.5 μl | 0.5 μM |
Template DNA | variable | variable | < 1,000 ng |
Nuclease-Free Water | to 25 μl | to 50 μl |
Notes: Two Primers have to be diluted 1:10 !
Notes: Gently mix the reaction. Collect all liquid to the bottom of the tube by a quick spin if necessary. Overlay the sample with mineral oil if using a PCR machine without a heated lid.
2. Transfer PCR tubes to a PCR machine and begin thermocycling.
4. Steps of PCR
2. Annealing : temperature is lowered to enable the DNA primers to attach to the template DNA.
3. Extending : temperature is raised and the new strand of DNA is made by the polymerases.
Thermocycling Conditions for a Routine PCR:
Step | Temperature | Time |
---|---|---|
Initial Denaturation | 98°C | 30 seconds |
98°C | 5–10 seconds | |
25–35 Cycles | *50–72°C | 10–30 seconds |
72°C | 20–30 seconds/kb | |
Final Extension | 72°C | 2 minutes |
Hold | 4-10°C |
Please note that protocols with Q5 High-Fidelity DNA Polymerase may differ from protocols with other polymerases. Conditions recommended below should be used for optimal performance.
[1] https://www.neb.com/protocols/2012/12/07/protocol-for-q5-high-fidelity-2x-master-mix-m0492 (accessed 31 October 2017)
1. Aim
2. Test digest - What for?
- targeted cuts between specific base sequences
- cleavage fragments small enough to run on gel
- segregation and analysis by gel electrophoresis
3. Preparative digest - What for?
4. Procedure
component | volume |
---|---|
DNA (typically 200 - 500 ng) | 1 µl |
10x buffer | 1 µl |
H2O | 7,6 µl |
enzyme 1 | 0,1 µl |
enzyme 2 | 0,1 µl |
final volume | 10 µl |
It is also possible to use more than two or just one restriction enzyme per reaction. They just need to have the same buffer preferences. Volumes have to be adjusted to the number of enzymes.
[1] http://www.log2embl.de/sites/default/files/Labor-Protokoll-Restriktionsverdau.pdf (accessed 30 October 2017)
[2] https://www.uni-hohenheim.de/fileadmin/einrichtungen/pflanzenphysiologie/Protokolle/V.Klonierung/restriktionsverdau_de.pdf (accessed 30 October 2017)
[3] http://www.biochemie.uni-jena.de/files/Praktikum/plasmid%20dna%20+%20restrictionsverdau.pdf (accessed 30 October 2017)
1. Aim
2. Helps us to pick the right colonies for colony-PCR and GC-MS measurements.
2. Safty
2. Reagent will always be mixed together on the spot, FeCl3 stock solution is finished, acid will be taken from the chemicals sheld from the AG plant physiology (has been negotiated).
3. What happens?
2. IAA will be seen as bright red (other compounds brown or yellowish).
4. Assay conditions
2. Plates are overlaid with Nitrocellulose immediately after inoculation with toothpicks. After normal incubation (i.e. overnight) time, the membrane was removed and soaked in reagent (or reagent-saturated [2.5 mL] filter paper, here “Whatman grade 2” had best results), in glass chamber ( danger symbol and written information).
3. After 30 - 60 minutes, coloring reaction is finished and fading began.
4. Best results with colony sizes between 0.5 to 2 mm.
5. Addition of Tryptophan greatly enhances color reaction but does not interfere with distinguishing IAA positive and negative colonies (yellow background and strong red to pink positives).
6. Other indolic compounds (i.e. indolepyruvic acid) are distinguishable by a more yellow-brownish color.
5. Afterwards
2. Throw away liquid and solid waste separately.
[1]Bric JM, Bostock RM, Silverstone SE. Rapid In Situ Assay for Indoleacetic Acid Production by Bacteria Immobilized on a Nitrocellulose Membrane. Applied and Environmental Microbiology. 1991;57(2):535-538. (accessed 31 October 2017)
1. Aim
2. Assembly of multiple DNA fragments into a recombinant DNA molecule → in a single in vitroreaction.
3. General principle: recombining short end homologies (15-52 bp.)
4. Homologous ends can be flanked by heterologous sequences (e.g. for inducing a linker sequence).
2. Steps
+ 50 μL 2 M MgCl2
+ 100 μL 100 mM ATP (Thermo #R0441)
+ 10 μL 1 M DTT
+ ddH2O to 1 mL
Store at -20 °C in 40-60 μl aliquots.
+ each additional assembly piece (1:1 - 1:10 molar ratio of vector:insert)
+ 1 μL 10X SLiCE buffer
+ 1 μL PPY SLiCE extract
+ ddH2O to 10 μL
-> Good to know: PPY in the strain whose cell extract is used for SLiCE reaction
4. Transform 1 - 10 μL of the assembly reaction into 50 μL of competent E. coli and/or run a diagnostic agarose gel to check for successful assembly.
Transformation of E. coli safer, but takes more time.
5. For electroporation, transform 1 μL into 50 μL electrocompetent cells. For large recombinant DNA, electroporation is required. In complex cloning, electroporation is recommended, as it is 10-100 times as efficient as chemical transformation.
Electrocompetent cells have to be made, or we use heat shock (see protocol“transformationof E. coli”), protocol for electrocompetent cells can be taken from NEB
[1] https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4672941/ (accessed 01 November 2017)
1. What is it?
2. Steps
2. Thaw a tube competent E. coli cells on ice for 10 minutes.
2. Pipette 50 µl of the cells into the 1.5 ml tube.
(as soon as as the last bit of ice in the tube is disappeared!)
4. Flick the tube 4-5 times to mix cells and DNA. (No vortexing!)
5. Place the mixture on ice for 30 minutes. (without mixing!)
(2-fold loss in transformation efficiency for every 10 minutes this step is shortened!)
6. Heat shock at exactly 42°C for exactly 30 seconds. (without mixing!)
(temperature and timing specific to transformation volume and vessel)
7. Place on ice for 5 minutes.
(without mixing!)
8. Pipette 950 µl of room temperature SOC into the mixture.
9. Place at 37°C for 60 minutes and shake vigorously (800 rpm in thermo mix block).
(2-fold loss in transformation efficiency for every 15 minutes this step is shortened)
(SOC gives 2-fold higher transformation efficiency than LB medium)
(incubation with shaking or rotating the tube gives 2-fold higher transformation efficiency than without)
10. Warm selection plates to 37 °C.
11. Mix the cells thoroughly by flicking the tube and inverting.
12. Spread 200 µl onto a selection plate and incubate overnight at 37 °C.
13.For low efficiency cloning reactions: spin down the whole transformation mixture and remove the nearly complete supernatant (approx. 900 µl). Resuspend cells in remaining liquid and plate completely.
[1]https://www.neb.com/protocols/1/01/01/high-efficiency-transformation-protocol-c2987 (accessed 01 November 2017)
1.Preparation of Competent Cells
(~5 x 106 - 2 x 107 cells/ml or OD 600 of 0.8-1.0).
The following steps are accomplished at room temperature.
3. Add 10 ml EZ 1 solution to wash the pellet. Repellet the cells and discard the supernatant.
4. Add 1 ml EZ 2 solution to resuspend the pellet. At this point, the competent cells can be used for transformations directly or stored frozen at or below -70°C for future use. It is important to freeze the cells slowly. To accomplish this, either wrap the aliquotted cells in 2-6 layers of paper towels or place in a Styrofoam box before placing in the freezer.DO NOT use liquid nitrogen to snap-freeze the cells.
2. Transformation
2. Incubate at 30 °C for 45 minutes. Mix vigorously by flicking with finger or vortexing (if appropriate for your DNA) 2-3 times during this incubation.
3. Spread 50-150 μl of the above transformation mixture on an appropriate plate. It is unnecessary to pellet and wash the cells before spreading. Incubate the plates at 30°C for 2-4 days to allow for growth of transformants.
[1] http://www.zymoresearch.com/downloads/dl/file/id/165/t2001i.pdf (accessed 01 November 2017)
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