Experiments
Molecular Biology (DNA)
BioBrick Assembly
If the vector is a plasmid there will generally be a region where the are multiple cloning sites. The insert does not typically have a cloning site built in unless they are left over from previous cloning. PRC can be used to generate a large amount and insert and add the appropriate cut sites.
Separately incubate both pieces of DNA (vector and insert) with two restriction enzymes. The vector and insert must be designed such that they have matching sticky ends. It is common to deactivate the restriction enzymes by heat denaturation once sufficient time had passed.
Now that both pieces of DNA have matching overhangs it is time to combine them and form the desired recombinant product.
Hopefully some of the recombinant products made their way into the cells. Antibiotic selction will filter out any cells that don't have a plasmid. Unfortunately, those plasmids could be empty...
The process of isolating DNA from a matrix. In our case it is either an E.coli cell lysate of an agarose gel.
Agarose Gel Electrophoresis separates out the DNA molecules by size. This protocol was placed at the end of the workflow, but in reality, it can move around a little. This is a very versatile technique, we used it for the following.
After DNA extraction you perform a small digest and run the result on a gel. This allows you to identify if the colony you extracted the DNA from actually has the desired recombinant product.
After PRC it is common to run the result on an agarose gel. Since the size of the PCR product is known you can see if the PCR reaction was successful. Furthermore, this serves as a method to purify the DNA as they will be separated out by size. Cut out the desired and extract the DNA from the gel.
Gibson Assembly
Gibson assembly relies on sequence complementarity between the peices of DNA being joined. Unless the DNA has been specifically preformatted you will need to add the complementarity to the 5' end by PCR.
The Gibson Reaction is an isothermal method: mix the following reagents, DNA with compatible overhangs, 5'-> 3' exonuclease, DNA polymerae and DNA ligase,
Molecular Biology (Proteins)
For studying the proteins alone one of the simplest options is the ITPG induced T7 polymerase system.
The most common method for protein purification is chromatography. By the use of a variety of stationary phases, proteins can be separated based on their size, hydrophobicity, isoelectric point or based on specific interaction. In our case, we used immobilized metal affinity chromatography (IMAC). Procedures below are for eGFP, but it is more or less the same with all His-tagged proteins.
Verification of Function
The file below describes the general procedure for both the phospate sensor and the nitrate sensro. The assay perfomed for the lehtbridge high shcool are very simmilar.
10X TAKEM4 buffer: 500 mM Tris pH 7.5,700 mM NH4Cl, 300 mM KCl, 10 mM EDTA, 40 mM MgCl2
The bioassays were all conducted by UNILausanne, the assay conditions are described below.