Team:UPF Barcelona/Experiments
This section consists in a recopilation of the protocols that we have used during the development of Hormonic. It also contains the notebook of both wetlab parts of the project: the intein-mediated T3 biosensor and the proof of concept.
EXPERIMENTS
Protocols
Experimentation is one of the fundamental parts related to our biosensor. The design of the experiments and also the adaptation of the protocols to each experiment allowed us to achieve the main objectives of our project. In this page you can find all the steps that we followed, because we want all the experiments to be repeatable and reproducible.
Description
This protocol uses the Branson Digital Sonifier 250 & Sonifier Sound Enclosure to lysate Bacterial E. coli Cells.
Description
Protocol for the digestion of DNA fragments for Biobrick assembly. It is necessary to prepare one Eppendorf for each DNA fragment that needs to be digested.
Description
Protocol for the preparation of electro-competent cells for the use of electroporation, a widely used method for transformation of DNA extracts into cells based on electric pulses.
Description
Electroporation is a widely used method for transformation of DNA extracts into cells. It is based on sending electrical pulses to the cell so its polarized membrane forms reversible transient pores that let the DNA flow into the cell. This protocol explains how to transform electro-competent cells via electroporation. This protocol is adapted from Woodall's work (Woodall, C. A. (n. d.). Electroporation of E. coli. E. coli Plasmid Vectors, 55–60. doi:10.1385/1-59259-409-3:55).
Description
Protocol for PCR using Kappa polymerase. The Kappa polymerase is a high-fidelity polymerase so it is a good option for DNA that needs to be transformed. It is necessary to prepare one tube for each PCR reaction. This protocol is for a 25 μL reaction.
Description
Protocol for the ligation of DNA fragments for Biobrick assembly. It is necessary to prepare in Eppendorfs a mix for every combination of DNA fragments that needs to be ligated.
Description
Protocol for PCR using Phusion polymerase. The Phusion polymerase is a high-fidelity polymerase so it is a good option for DNA that needs to be transformed. It is necessary to prepare one tube for each PCR reaction. This protocol is for a 20 μL reaction.
Description
Protocol for the digestion of DNA fragments for running a gel to check if an insert is present. It is necessary to prepare in Eppendorfs a mix for every combination of DNA fragments that we want to digest.
Description
Protocol for a colony PCR using Taq polymerase. The Taq polymerase is not a high-fidelity polymerase so it is a good option for checking if a colony has an insert. It is necessary to prepare one tube for each PCR reaction. This protocol is for a 25 μL reaction.
Description
Protocol for the transformation of DNA via heat shock using the DH5α strain of E. coli. It is necessary to prepare one Eppendorf for each DNA fragment that needs to be transformed.
Description
Protocol for Western blot procedure and the sample preparation. With this technique we can detect the presence of the desired protein. In order to quantify it, we have to apply the SDS-PAGE protocol.
T3 biosensor notebooks
Experimentation is key in Synthetic Biology. Even though the design can be based on literature and theory, biology never reacts exactly as you planned, and that’s a fact. So, the only way to prove the design of our intein-mediated T3 (IMT3) biosensor was to actually bring it to reality. Here, we present the Benchling notebooks we used to track the different experiments we did to make our sensor come true.
E. coli DH5α Transformation: Here we explain how we used E. coli DH5α cells as DNA storage.
E. coli BL21 Transformation: Here we explain how we prepared E. coli BL21 (DE3) to be electro competent and how we transformed them to express both of our IMT3 biosensors as our main chassis for its good recombinant protein expression.
Western Blots: Here we have described the different Western Blots we did to check the correct expression and splicing of our protein.
Plate-readers: This notebook describes the preparation of our strains and wells content to sense in a Plate-reader over the course of long time periods and see its reaction with different T3 concentrations. As expected, we had some biology related problems, but technology was not in our side all the time either.
Proof of concept notebook
As a Proof of Concept for our biosensor we decided to build a lactone biosensor. This notebook contains the different cloning strategies performed to build the genetic construct and transform it into our sensor cells. Once the sensor was complete, a full characterization of its performance was done, using both, fixed concentrations of lactone and our lactone producer cells to determine the dynamic range of our lactone sensor cells. This notebook also contains all the wetlab experiments performed to do these characterizations.
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