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Team:Valencia Biocampus/ResultsDemoTestATOPE
Our Results
Stability
Escherichia coli is the lab rat of the bacterial world.
We have performed the most complete characterization of the stability of E. coli in order to both confirm its optimality as a chassis for Synthetic Biology, and to determine the behavior of Biobricks under sub-optimal environmental conditions. We have subjected wild type strains to a range of stressful conditions (see just below "the limits of E. coli") and measured how our Biobricks behaved.
Standardization
Orthogonality results
In Synthetic Biology, two constructions can be considered orthogonal when they only interact at specific and predictable interphases, and do not disturb
each other. We have studied this desirable feature of Biobrick parts by combining two constructions in the same cell and comparing this output with the one
produced by simple transformations. To do this, we used both standard fluorimetry assays and flow cytometry, which allowed to get information at the cell level. Then, we wanted to go one step further: what is the effect of a simple transformation (a plasmid with a Biobrick part) into the cell architecture? To get some insights about this,
we performed a proteomic analysis in which the whole proteome of an E. coli strain transformed with a Biobrick part was
compared to that of the non-transformed, control strain. Last, but not least, a detailed set of equations modeling the behavior of cells carrying two Biobrick parts was developed.
1. Standard Fluorimetry assays
We performed experiments with two different combinations of Biobrick parts containing a fluorescent protein under the control of a promoter sequence from
Anderson’s promoter collection: Bb1 (GFP with strong promoter J23104) + Bb2 (RFP with strong promoter J23104) and Bb2 + Bb3 (GFP with less strong promoter
J23110). In all cases, several controls were used: wild type (non-transformed) cells, simple transformations with either the biobrick or an empty plasmid,
and co-transformations of the biobricks and the empty plasmids. It has to be noted that there was a high background emission of red fluorescence by the cells in all cases. Our results are shown in Figures 1 and 2.
General protocol(1)
Open License (and Responsible Research and Innovation)
Not unlike scientific research, RRI is about common sense, about combining self-profit with others’ profit, about taking care of our planet. We believe that being responsible is not the opposite of being selfish; responsibility is just a broad-minded way of selfishness that points at the long-term benefit.
And there is no long-term benefit without a sustainable society in a sustainable environment. What is good for our neighbors and for our home must be good for us as well.
Our Proposal: Responsible Research and Innovation as a tool to choose within the IP ecosystem.
Since we, our Human Practices sub-team, are a biotechnology and a law student, we are also glad to see that another key concept in IP is a beautiful science-law metaphor: Jane Calvert & Drew Endy's “diverse ecology” scenario. This image fits well with our interdisciplinary spirit and we found it very useful because it evokes very elegantly the range of different solutions out there to choose from in order to use a scientific invention in a given way.
The seat of the ST2OOL
One of the goals of our project was to select standard, stable, and orthogonal parts naturally occurring in nature. To do that, we carried out a functional
metagenomics strategy aiming at selecting promoters from a library of metagenomic DNA. As a first step, we wanted to isolate metagenomic DNA from
environmental samples, but instead of using traditional manual kits, we decided to build a robot able to automatically extract metagenomic DNA for us… It
is a pleasure for our team to introduce you our TOOL robot:
Modeling
We summarize here our main contributions modeling The ST$^2$OOL. Our hypothesis, formulae and analysis can be found in the modeling section.
Stability
Escherichia coli is the lab rat of the bacterial world. We have performed the most complete characterization of the stability of E. coli in order to both confirm its optimality as a chassis for Synthetic Biology, and to determine the behavior of Biobricks under sub-optimal environmental conditions. We have subjected wild type strains to a range of stressful conditions (see just below "the limits of E. coli") and measured how our Biobricks behaved.
Standardization
Orthogonality results
In Synthetic Biology, two constructions can be considered orthogonal when they only interact at specific and predictable interphases, and do not disturb each other. We have studied this desirable feature of Biobrick parts by combining two constructions in the same cell and comparing this output with the one produced by simple transformations. To do this, we used both standard fluorimetry assays and flow cytometry, which allowed to get information at the cell level. Then, we wanted to go one step further: what is the effect of a simple transformation (a plasmid with a Biobrick part) into the cell architecture? To get some insights about this, we performed a proteomic analysis in which the whole proteome of an E. coli strain transformed with a Biobrick part was compared to that of the non-transformed, control strain. Last, but not least, a detailed set of equations modeling the behavior of cells carrying two Biobrick parts was developed.
1. Standard Fluorimetry assays
We performed experiments with two different combinations of Biobrick parts containing a fluorescent protein under the control of a promoter sequence from Anderson’s promoter collection: Bb1 (GFP with strong promoter J23104) + Bb2 (RFP with strong promoter J23104) and Bb2 + Bb3 (GFP with less strong promoter J23110). In all cases, several controls were used: wild type (non-transformed) cells, simple transformations with either the biobrick or an empty plasmid, and co-transformations of the biobricks and the empty plasmids. It has to be noted that there was a high background emission of red fluorescence by the cells in all cases. Our results are shown in Figures 1 and 2.
General protocol(1)
Open License (and Responsible Research and Innovation)
Not unlike scientific research, RRI is about common sense, about combining self-profit with others’ profit, about taking care of our planet. We believe that being responsible is not the opposite of being selfish; responsibility is just a broad-minded way of selfishness that points at the long-term benefit. And there is no long-term benefit without a sustainable society in a sustainable environment. What is good for our neighbors and for our home must be good for us as well.
Our Proposal: Responsible Research and Innovation as a tool to choose within the IP ecosystem.
Since we, our Human Practices sub-team, are a biotechnology and a law student, we are also glad to see that another key concept in IP is a beautiful science-law metaphor: Jane Calvert & Drew Endy's “diverse ecology” scenario. This image fits well with our interdisciplinary spirit and we found it very useful because it evokes very elegantly the range of different solutions out there to choose from in order to use a scientific invention in a given way.
The seat of the ST2OOL
One of the goals of our project was to select standard, stable, and orthogonal parts naturally occurring in nature. To do that, we carried out a functional metagenomics strategy aiming at selecting promoters from a library of metagenomic DNA. As a first step, we wanted to isolate metagenomic DNA from environmental samples, but instead of using traditional manual kits, we decided to build a robot able to automatically extract metagenomic DNA for us… It is a pleasure for our team to introduce you our TOOL robot:
Modeling
We summarize here our main contributions modeling The ST$^2$OOL. Our hypothesis, formulae and analysis can be found in the modeling section.
