Team:Goettingen/Project/Methods

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Agarose Gel Electrophoresis

For the analysis of PCR-amplified products, agarose gel electrophoresis is the method of choice. This method takes advantage of the separation of DNA in dependance of the charge-mass ratio. The separation is based on the electric attraction of the negative charged DNA which is guided towards the positive charged anode upon application of a current. The PCR samples are run on agarose gels with different percentages according to the product sizes: small products run faster than bigger products. Later on, these fragments within the gel are made visible by examination under the UV light to ensure the correct DNA fragment length synthesized in the PCR reaction. Prior to UV analysis, a staining method of the DNA, here using ethidium bromide (EtBr), is obligatory. EtBr is an intercalating agent which embeddes itself within the DNA helix. Thus, the absorption spectrum is biased so that it is suitable for DNA detection. The determination of separated molecule sizes is done accodrding to a common DNA size standard.

Pouring the Gel
1% agarose gels are standard to separate DNA. The percentages and thus the degree of polymerisation of the gel influences the degree of separation, i.e. PCR products of similar size can be distinguished by applying a percentage lower than 1%. To pour a 1% agarose gel weight 1 g of Ultra Pure Agarose for every 100 ml of 1x TAE buffer. For preparation of 1x TAE buffer fill 10 mL of 50x TAE buffer stock ad 500 mL ddH₂O. Ensure that the 1% agarose in 1x TAE buffer is boiled throughly and dissolved completely without streaks. Incomplete boiling will bias the separation results. The liquid is poured in a gel tray with the wanted comb which are assembled in a holder. After solification the gel is placed in a gel chamber and is fully covered with 1x TAE buffer post removal of the comb.

Loading the Gel
Prior to loading, the DNA samples are homogenized with 6x Loading Dye (LD) resulting in a 1x final concentration of the LD (4 μl 6x LD ad 20 μl PCR reaction). Be aware of the fact, that PCR samples should not be mixed with LD if further experiments with the samples are needed to be done. If this is the case, mix the PCR samples with LD on parafilm. Meanwhile, the PCR samples are kept on ice. The 1 kb DNA ladder is stored in the 4°C fridge and is suitable for larger PCR products ranging from 250 bp to 10000 bp (GeneRuler 1 kb DNA Ladder (Fermentas)). Load the gel with x μL of LD-mixed samples next to the first well with 5 μl marker. The amount loaded for PCR samples depends on the concentration and the size of the products. Note that PCR tubes should be closed to prevent drying up exposed to air.

Running the Gel
When all samples have been loaded, connect the power supply. Ensure that the plus pol is at the opposite site of the loaded wells. Run big gels applying 100 V for a more accurate separation for about 1 h. The time may be adjusted according to the loading front.

Staining the Gel
To make the separated DNA bands visible under the UV light, the samples were stained in a EtBr bath for about 10 min. Then, the gel was rinsed in water for approximately 5 min. The EtBr stained gel was exposed to UV light under a gel documentation station, a gel photo was taken and saved under the iGEM2012 folder. The file should be labelled with the date, group number and short description of what was analyzed. A printed version should be pasted into the group notebook.

Cloning Protocols

Cloning Protocols: Ligation

Combination of 50 ng of vector with a 2-fold molar excess of insert.
Addition of an appropriate volume of T4 Buffer.
Addition of 0.5-1 μl T4 ligase.
Adjustment of volume to 20 μl with ddH₂O
Incubation over night at 16°C
Deactivation of ligase for 10 min at 65°C

Cloning Protocols: Chemical Transformation

Thawing of competent cells on ice
Addition of 1 μl DNA
Incubation on ice for 20 min
Heat shock for 1 min at 42°C
Incubation on ice for 5 min
Addition of 270 μl LB
Incubation in thermoblock for 45 min (incubation time dependent on used vector!) at 37°C and 300 rpm
Smearing of cells on plates and incubation at 37°C (centrifuge it, discard supernatant, resuspend cells in rest-medium, 20 μl on plate)

Competent Cells

Preparation of CaCl₂ buffer for competent cells! Before you start make sure that the CaCl₂ buffer is ice-cold when needed and the centrifuge is cooled to 4°C.

Inoculate LB-liquid medium with 1 ml of overnight culture.
Let cells grow to OD₆₀₀ 0.4-0.6.
Transfer them to 50 ml Falcons and centrifuge for 5 min at 1500 g and 4°C.
Resuspend pellet in 5-10 ml CaCl₂ buffer; here 7 ml (prechilled).
Centrifuge 5 min 1500 g, 4°C
Resuspend pellet in 5-10 ml CaCl₂ buffer; here 7 ml (prechilled).
Incubate cells 30-60 min on ice; here 30 min; Eppis prechilled in 5-10 ml on dry ice (or in liquid nitrogen)
Storage at -80°C

Library Selection

The library containing vectors with the mutagenized tar-gene was transformed into the E. coli strain Bl21. In order to determine certain receptor derivates that enables chemotaxis to a certain molecule a "Library Selection" protocol was determined.
Attractants
Coffein: stock solution x mM
2-Ethyl-1-Hexanol: stock solution x mM
Geraniol: stock solution x mM
Sodium-Cyclamat: stock solution x mM
D-aspartic acid: stock solution x mM
L-aspartic acid 4-benzyl: stock solution x mM
Vanillin: stock solution x mM

Media
- LB-media
- Tryptone-agar (1% tryptone, 0.5% NaCl, 0.3% agar)
Execution
First round of selection
- One 1 ml cryostock of the library in Bl21 was thawed and poured into a 200 ml flask filled with LB-media with chloramphenicol
- The BL21 strain with the parent plasmid was inoculated in 5 ml LB media with chloramphenicol - The cultures were grown over night at 37 °C with approx. 180 rpm
- 7 + 1 control (whatmanpaper with H2O) 12 cm petridishes were filled with 0.3% tryptone-swimming agar with chloramphenicol
- 100µl of the attractant were applied to a steril 2x2cm whatmanpaper respectivly and positioned in the center of a petridish
- At least 1.5 ml of the culture containing the library and at least 1.5 ml of the culture containing the BL21 strain with the parent plasmid were spun down with 1.5 X g for 10 minutes
- The supernatant was discarded and the pellet was resuspended in the remaining medium
- 3 times 5 µl of the library and once 5 µl of the reference strain (Bl21 with the parent plasmid) were dropped respectively on each plate
- The drops were dried for at least 20 minutes until the plates were inverted and placed in the incubator at 33°C over night

Second round of selection
- On each plate the drop with the fastes and most directed swimming behaviour was determined
- In order to select the fastest cells the cells containing agar were cut out:

- the yellow eppendorf tips were cut of to the first mark (approx 1 cm)

- the first cut off was shortly befor the swimming front --> I

- the second cut off was on the swimming front -->II

- the third cut off was shortly behind the swimming front --> III

- each cut off was placed either with or without the tip in at least 0.5 ml LB media in an test tube or an E-cup

- The cultures were incubated for at least 1 h at 37 °C with approx. 180 rpm
- Meanwhile 7 + 1 control (whatmanpaper with H2O) 12 cm petridishes were filled with 0.3% tryptone-swimming agar with chloramphenicol
- 100µl of the attractant were applied to a steril 2x2cm whatmanpaper respectivly and positioned in the center of a petridish
- The cultures were transferred into an E-cup and were spun down with 1.5 X g for 10 minutes
- The supernatant was discarded and the pellet resuspended in the remaining medium
- 5 µl of the 3 different library cut offs and 5 µl of the reference strain (Bl21 with the parent plasmid) were dropped respectively on each plate
- The drops were dried for at least 20 minutes until the plates ware inverted and placed in the incubator at 33°C over night

Third round of selection
- See second round of selection

Plating of the selected clones
- The plates of the third round of selection were treated as described before, but the cultures are not spun down
- 100 µl of a 10^-2 dilution was plated on LB-plates containing chloramphenicol respectively
- The plates were incubated in an incubator over night at 33 °C

Minipreparation and sequencing of plasmid DNA
- A suitable amount of clones was selected from each plate and used to inoculate 5 ml LB media with chloramphenicol respectively
- The cultures were incubated over night at 37 °C with approx. 180 rpm
- The plasmid DNA was isolated according to the instructions of the the peqlab kit
- The plasmid DNA was sequenced as described

Retransformation of the plasmid DNA
In order to determine wether the observed chemotaxis was dependent on the cells themselfes or on the inserted vector the isolated plasmid DNA was transformed into fresh BL21 cells according to the described protocol

Determination of the swimming behaviour of the freshly transformed BL21 cells
- Colonies of the freshly transformed BL21 cells (Retrafo) as well as of the selected BL21 clones (Trafo) were inoculated in LB-media with chloramphenicol and grown over night at 37 °C with approx. 180 rpm - 7 x 2 x 3 0.3% tryptone-swimming agar plates were poured

- Each attractant had 2 additional controls: one time the whatmanpaper was soaked with H2Odest. and the other time with aspartate

- The whole approach was conducted for the "Trafos" as well as for the "Retrafos"

- The cultures were treated and dropped on the plates as described
- The drops were allowed to dry for at least 20 minutes until the plates were inverted and placed in the 33°C incubator over night

Sequencing

Under process...

Standard PCR

The polymerase chain reaction (PCR) is a method for in vitro-amplification of DNA sequences. For the amplification of a DNA fragment the heat resistent enzyme DNA polymerase is responsible. There are several types of DNA polymerases purchaseable, e.g. some of which are very fast or are not error-prone due to proof-reading activity. In order to choose the appropriate DNA polymerase, this link might be of interest: http://barricklab.org/twiki/bin/view/Lab/ProtocolsTaq; 06/30/2012.
To allow binding of the DNA polymerase primer are required. Thus, only the flanking sites for the sequence of interest needs to be known to synthesize specific primer. Each primer has a specific annealing temperature according to it GC-content. Primers used here were ordered by Sigma^®. A single PCR cycle encompasses three steps: denaturation, annealing and elongation. In the first step the DNA double strands are separated at about 95°C. Next hybridization of the primers at their annealing temperature happens and finally the DNA seqthesis takes place at the optimal working temperature of the chosen DNA polymerase. This amplification cycle is normally conducted between 25-35 cycles depending on the amount of PCR product one wants to synthesize. In one PCR reaction following components are mixed: DNA template, forward and reverse primer, DNA polymerase with appropriate buffer and deoxynucleotides. Note that an increasing cycle number is prone to incorporate errors in the amplified DNA fragment. This is due to the fact that the dNTPs will be depleted and the saturation phase is reached.

QuikChange Protocol

To remove disturbing restriction sites within the gene for the successful usage of BioBrick system, the QuikChange reaction is used.

-> 20 μl / reaction:

2 μl 10x Buffer
0.4 μl dNTPs (10 mM each)
0.8 μl Primer (10 µM) (2x or as premix)
0.4 μl Template (ca. 20-100 ng)
0.4 μl Pfu-Turbo
ad 20 μl ddH2O

Program: -> 1 min 96°C

20 s 96°C
20 s 58°C
1.5 min/kb 72°C
15 Cycles

-> 5 min 72°C -> Store at 4-8°C

After PCR add 1 μl DpnI directly into PCR tube. Incubate reaction 1-2 h at 37°C. Transform 5 µl into 50 µl competent cells.

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