Difference between revisions of "20.109(F11): Mod 2 Day 3 Tools for system engineering"
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==Introduction== | ==Introduction== | ||
[[Image:EvolutionBiolSystem.png|thumb|left]]Biological systems are not static. Thus they can be engineered to account for changing environmental conditions, as we've seen through our examination of two component regulatory systems. In addition, they can be engineered to account for changes that occur as the cells replicate and divide over time. Indeed, evolution of biological systems away from an original specification can be viewed as a curse (it's not like computer scientists have to worry that their software programs change functions when they relaunch them!) or a blessing (evolution can find a solution we didn't ever dream of). Here we'll take the rosy view and try to harness genetic variability to improve the bacterial photography system. In particular we'll screen a library of changes in the Cph8 gene to find ones that increase the phosphatasing activity of the sensor when the cells are growing in the light. The mutants (we'll call them P+) should make the "light" color of the photographs more light by decreasing the amount of phosphorylated OmpR that stimulates LacZ transcription. | [[Image:EvolutionBiolSystem.png|thumb|left]]Biological systems are not static. Thus they can be engineered to account for changing environmental conditions, as we've seen through our examination of two component regulatory systems. In addition, they can be engineered to account for changes that occur as the cells replicate and divide over time. Indeed, evolution of biological systems away from an original specification can be viewed as a curse (it's not like computer scientists have to worry that their software programs change functions when they relaunch them!) or a blessing (evolution can find a solution we didn't ever dream of). Here we'll take the rosy view and try to harness genetic variability to improve the bacterial photography system. In particular we'll screen a library of changes in the Cph8 gene to find ones that increase the phosphatasing activity of the sensor when the cells are growing in the light. The mutants (we'll call them P+) should make the "light" color of the photographs more light by decreasing the amount of phosphorylated OmpR that stimulates LacZ transcription. | ||
− | The region of the Cph8 protein to focus on for this purpose has been defined through traditional scientific studies of EnvZ, for example the work from Tom Silhavy's lab( [ | + | The region of the Cph8 protein to focus on for this purpose has been defined through traditional scientific studies of EnvZ, for example the work from Tom Silhavy's lab( [http://openwetware.org/wiki/PMID:_9721293 ] and [[Media:K+P- JBact98.pdf|pdf]] here). We've also been guided by the expertise of MIT's [http://web.mit.edu/biology/www/facultyareas/facresearch/laub.html Mike Laub,] whose lab studies the specificity and rewiring of two component regulatory systems. From these sources, a span of 5 contiguous amino acids can be identified as relevant for shifting the balance of EnvZ to greater kinasing or greater phosphatasing activity. These five residues in EnvZ are Alanine at amino acid 239 ("A239") through Histidine at amino acid 243 ("H243"), where mutations in the flanking residues (A239 and H243) have been shown to enhance the phosphatase activity of EnvZ and mutations in the internal residues (G240 V241 S242) enhance the kinase activity of EnvZ. The amino acid changes that modify the enzymatic activities are indicated on the figure below. Two important notes about these mutations though: First, the balance of kinase to phosphatase activities have been affected by the changes, but the mutations do not shift the reactions to fully "on" or fully "off." Second, the fusion protein of Cph1 to EnvZ, called Cph8, changes the numbering of the residues, as shown in the figure below. It's hoped, however, that the local environment of the region is similar to the natural EnvZ protein. [[Image:P+, EnvZ, Cph8 align.png]]<br> |
To complement the genetic approach for solving biological engineering puzzles, we'll also consider three more approaches in synthetic biology. The first is a [http://www.partsregistry.org/Main_Page Registry of Standard Biological Parts,] essentially a community resource that has some ready-made and useful genetic elements that can be assembled into synthetic biological devices systems. The second approach is to recapitulate the genetic network of the biological system using electronic components, making explicit some of the benefits and limitations of such an approach and the often-cited analogy between building with biology and building with computer programs or electronic components. The final approach is the one you started last time, namely the use the [http://www.tinkercell.com/ Tinkercell computer model] to simulate the behavior of the bacterial photography system, with the goal of performing "in silico" experiments that would take days or weeks to do at the bench. | To complement the genetic approach for solving biological engineering puzzles, we'll also consider three more approaches in synthetic biology. The first is a [http://www.partsregistry.org/Main_Page Registry of Standard Biological Parts,] essentially a community resource that has some ready-made and useful genetic elements that can be assembled into synthetic biological devices systems. The second approach is to recapitulate the genetic network of the biological system using electronic components, making explicit some of the benefits and limitations of such an approach and the often-cited analogy between building with biology and building with computer programs or electronic components. The final approach is the one you started last time, namely the use the [http://www.tinkercell.com/ Tinkercell computer model] to simulate the behavior of the bacterial photography system, with the goal of performing "in silico" experiments that would take days or weeks to do at the bench. | ||
==Protocols== | ==Protocols== | ||
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#Hold the pulse button until you hear a beep. Listen carefully since the beep is not loud. | #Hold the pulse button until you hear a beep. Listen carefully since the beep is not loud. | ||
#'''Quickly''' remove the cuvette from the holder and '''immediately''' add the 0.5 ml volume of "SOC" media to the cells. Delaying this addition by even 1 minute has been seen to decrease transformation by 3 fold. | #'''Quickly''' remove the cuvette from the holder and '''immediately''' add the 0.5 ml volume of "SOC" media to the cells. Delaying this addition by even 1 minute has been seen to decrease transformation by 3 fold. | ||
− | # Transfer the cells and the media back to an eppendorf tube and place the tubes on the nutator in the | + | # Transfer the cells and the media back to an eppendorf tube and place the tubes on the nutator in the 37 incubator for 1 hour. During this incubation you can work on Parts 2, 3 and 4 of today's protocols. |
− | # Spread 20 ul of the electroporation mix onto a Tetrazolium+Cam34+Amp25+Kan10 petri dishes. Plate 200 ul of the electroporation mix on another Tetrazolium+Cam34+Amp25+Kan10 petri dish. One of these two dilutions should have single, well-isolated colonies to examine next time. Incubate the plates at | + | # Spread 20 ul of the electroporation mix onto a Tetrazolium+Cam34+Amp25+Kan10 petri dishes. Plate 200 ul of the electroporation mix on another Tetrazolium+Cam34+Amp25+Kan10 petri dish. One of these two dilutions should have single, well-isolated colonies to examine next time. Incubate the plates at 37 in the light until next time. |
===Part 2: Registry of Standard Biological Parts=== | ===Part 2: Registry of Standard Biological Parts=== | ||
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The analogy of the DNA as computer code is not perfect. We have to set aside the presumption of an intelligent agent responsible for writing the initial program as well as accept that natural events will change the code over time (evolution leading to genetic variation--the very thing we're trying to harness in the first part of today's lab). And no good tools exist for systematically debugging the genetic code. | The analogy of the DNA as computer code is not perfect. We have to set aside the presumption of an intelligent agent responsible for writing the initial program as well as accept that natural events will change the code over time (evolution leading to genetic variation--the very thing we're trying to harness in the first part of today's lab). And no good tools exist for systematically debugging the genetic code. | ||
− | What would make genetic code easier to write? One idea is to make it a more | + | What would make genetic code easier to write? One idea is to make it a more |