Team:UPF Barcelona/Protocols notebook
Wetware
Protocols and notebook
Protocols
The protocols have guided us in developing the experiments in the laboratory. Before starting each experiment we searched for the proper protocol. Then, the laboratory team met and studied the materials and techniques necessary to carry out the whole process and adapt it to our specific experiment. In this part, we are going to present the different protocols that we have used in our project divided into distinct modules corresponding to each specific wet lab development processes.
1. Backbone plasmid modifications
Bacterial liquid culture
Used for growing large amounts of bacteria. Usually kept overnight at 37ºC to give bacteria time to grow.
Miniprep and OD measurement
Used for DNA isolation. The success of this procedure is checked with an optic density (OD) measurement.
PCR amplification
For plasmid modification a PCR was performed, in order to selectively amplify the regions of interest.
Agarose gel electrophoresis
Running the PCR results in an agarose gel gives information about the replicons’ size, and thus about the success of the procedure.
Digestion with DpnI
Digestion with this restriction enzyme will degrade the DNA template used in the PCR amplification. This way, only the amplicon will remain intact.
DNA purification (clean-up)
This procedure allows for removing residual DNA from previous reactions. A commercial kit is used for this purpose.
Phosphorylation of the linear DNA ends
This protocol must be performed in order to have the necessary phosphate groups on each end of the DNA molecule.
DNA ligation
Allows for joining both ends of the linear DNA molecule to obtain a circular plasmid backbone.
2. NYZ5α competent cells transformation
Heat-shock transformation of NYZ5α competent cells
Transformation of this bacterial strain will allow to obtain increased copies of the plasmid, since it is engineered to achieve this goal.
Colony PCR
PCR directly on the bacterial colonies isolated from a plate. Will help to verify if the plasmid was correctly incorporated.
Agarose gel electrophoresis
Running the PCR results in an agarose gel gives information about the replicons’ size, and thus about the success of the procedure.
Sequencing
Prior to sending the DNA product to a sequencing service, a reaction must be prepared with all the necessary components for this process.
3. Plasmid cloning (LbCas12a, specific gRNAs)
Bacterial liquid culture
Used for growing large amounts of bacteria. Usually kept overnight at 37ºC to give bacteria time to grow.
Miniprep and OD measurement
Used for DNA isolation. The success of this procedure is checked with an optic density (OD) measurement.
ssDNA phosphorylation and annealing
ssDNA oligonucleotides, which are synthetically constructed, must be phosphorylated and annealed to obtain a dsDNA insert suitable for cloning.
Golden Gate Assembly
Allows for cloning the chosen plasmid with the dsDNA fragment previously constructed.
4. Transformation of the vectors into BL21 competent cells
Electroporation transformation into BL21 competent cells
Transformation of this bacterial strain will allow to obtain increased protein expression, since it is engineered to achieve this goal.
Colony PCR
This procedure consists of performing a PCR directly on the bacterial colonies isolated from a plate. Will help to verify if the plasmid was correctly incorporated.
Agarose gel electrophoresis
Running the PCR results in an agarose gel gives information about the replicons’ size, and thus about the success of the procedure.
Sequencing
Prior to sending the DNA product to a sequencing service, a reaction must be prepared with all the necessary components for this process.
5. Biosensors testing
IPTG induction of the plasmids expression
Adding this monosaccharide to the culture media will indirectly activate transcription of the genes modulated by a T7 promoter.
Cell lysis
Leads to releasing of the cell’s internal components. Three approaches: mechanical lysis, enzymatic lysis (with and without EDTA).
gRNA-Cas12a assay and fluorescence read-out
Cleavage of the template DNA with its corresponding gRNA-Cas12a and determination of the biosensors efficiency using a commercial kit reporter.
6. In vitro gRNA-Cas12a assay
PCR
In this case, a PCR amplification was performed to obtain greater amounts of the DNA aiming to be transcribed.
HindIII digestion
Shortening of the DNA template to achieve an optimal gRNA length, since gRNA plasmid designs lacked a functional transcription terminator.
DNA purification (clean-up)
This procedure allows for removing residual DNA from previous reactions. A commercial kit is used for this purpose.
Agarose gel electrophoresis
Running the DNA product from the previous step in an agarose gel will give information about its final length.
In vitro transcription
This process will allow transcribing the DNA template into potentially functional RNA.
RNA isolation
After the in vitro transcription, the final product must be isolated from the other reaction components.
In vitro gRNA-Cas12a digestion
Cleavage of the dsDNA template (sample) with its corresponding gRNA-Cas12a.
Fluorescence read-out
Determine the efficiency of the constructed biosensors using a commercial kit reporter.
7. Autolysis plasmid cloning
PCR amplification
To add overhangs to the gene fragment containing the autolysis protein (Protein E).
Digestion with NheI and PstI
These restriction enzymes will open the backbone plasmid for cloning into it the DNA insert.
Agarose gel electrophoresis
Running the DNA product from the previous step to verify that both the PCR and the Digestion have worked.
Gibson assembly
This procedure was used to carry out the autolysis protein (Protein E) cloning into a pBAD-AraC plasmid backbone.
Heat-shock transformation into NZYalpha cells
Transformation of this bacterial strain will allow to obtain increased copies of the plasmid, since it is engineered to achieve this goal. Cells must then grow in the presence of glucose (See results).
Colony PCR
This procedure consists of performing a PCR directly on the bacterial colonies isolated from a plate. Will help to verify if the plasmid was correctly incorporated, after running an electrophoresis gel and sequencing the amplified fragment.
Induction with L-Arabinose
The addition of this reagent will trigger the autolysis activity of protein E and lysate the bacterial cells.
Bradford protein assay
This method is used to quantify the amount of protein that is released by the lysate cells.
8. Other complementary procedures
LB Agar Plate preparation
These will serve for growing colonies of the modified or transformed bacterial strains.
LB media preparation
Preparation of LB batches is essential for rapid preparation of liquid cultures.
Glycerol stock for plasmid preservation
In order to freeze transformed cells with a plasmid of interest for a long time, it is necessary to prepare a glycerol solution for cryopreservation.
Lab notebook
The lab notebook has been very useful for us to write down the experiments and the way in which they had been carried out. It has been a very useful tool for all the lab team to be aware of the protocols performed during the day and to learn how to improve them. We used it as a daily diary, and it was key for error detection and wet lab team coordination. Complementary, we also used an excel file to keep track of our reagents and plasmids locations and availability.
