Difference between revisions of "20.109(F09): 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 either increase the kinasing activity of the sensor when the cells are growing in the dark OR increase the phosphatase activity of the sensor when the cells are growing in the light. The first class of mutants (we'll call them K+) should make the "dark" color of the photographs more dark by increasing the amount of phosphorylated OmpR that stimulates LacZ transcription. The second class of mutants (we'll call these P+) should make the "light" color of the photographs lighter by decreasing the cell's levels of phosphorylated OmpR, thereby decreasing 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 either increase the kinasing activity of the sensor when the cells are growing in the dark OR increase the phosphatase activity of the sensor when the cells are growing in the light. The first class of mutants (we'll call them K+) should make the "dark" color of the photographs more dark by increasing the amount of phosphorylated OmpR that stimulates LacZ transcription. The second class of mutants (we'll call these P+) should make the "light" color of the photographs lighter by decreasing the cell's levels of phosphorylated OmpR, thereby decreasing 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:EnvZ,Cph8 align.png]]<br> |
To complement the genetic approach for solving biological engineering puzzles, we'll also consider two other 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 model biological systems, in this case we'll recapitulate the genetic structure of the bacterial photography system using electronic components, making explicit some of the benefits and limitations of such an approach. | To complement the genetic approach for solving biological engineering puzzles, we'll also consider two other 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 model biological systems, in this case we'll recapitulate the genetic structure of the bacterial photography system using electronic components, making explicit some of the benefits and limitations of such an approach. | ||
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===Part 1: Library Screen=== | ===Part 1: Library Screen=== | ||
The details for how the libraries were constructed and what kinds of changes are reasonable to expect will be considered in the next lab session. For today, you will transform a pool of DNA with degeneracies in the positions that affect the kinasing or phosphatasing activity of EnvZ. The recipient bacterial strain is identical to the bacterial photography system except that it does not harbor a plasmid encoding the light-sensing fusion protein Cph8. It does encode the OmpR-regulated LacZ gene as well as the phycobillins from a plasmid. | The details for how the libraries were constructed and what kinds of changes are reasonable to expect will be considered in the next lab session. For today, you will transform a pool of DNA with degeneracies in the positions that affect the kinasing or phosphatasing activity of EnvZ. The recipient bacterial strain is identical to the bacterial photography system except that it does not harbor a plasmid encoding the light-sensing fusion protein Cph8. It does encode the OmpR-regulated LacZ gene as well as the phycobillins from a plasmid. | ||
− | [[Image:Macintosh HD-Users-nkuldell-Desktop-20.109(F07)-20.109(F07) Mod3 ECD-Mod3F07 wiki images-micropulser.JPG|thumb]]To carry out the transformation you will try your hand at a different technique for getting DNA into cells, namely electroporation. This method involves exposing a small but dense volume of cells to a pulse of current. The pulse momentarily flips the lipid bilayer, opening small spaces for the DNA to pass into the cells. As you can imagine, the cells aren't fond of such treatment and the single most important step to help them recover is to '''quickly add media to the cells''' once they've been electroporated. The process is also very sensitive to salts in the DNA, and if you pipet too much DNA to the cells, the extra salt may cause an electrical "arc," (you'll know this has happened from the flash of light and the "pop" you'll hear) frying the cells dead. If this happens, please get another aliquot of cells from the faculty and try the electroporation again, with less DNA. | + | [[Image:Macintosh HD-Users-nkuldell-Desktop-20.109(F07)-20.109(F07) Mod3 ECD-Mod3F07 wiki images-micropulser.JPG|thumb]]To carry out the transformation you will try your hand at a different technique for getting DNA into cells, namely electroporation. This method involves exposing a small but dense volume of cells to a pulse of current. The pulse momentarily flips the lipid bilayer, opening small spaces for the DNA to pass into the cells. As you can imagine, the cells aren't fond of such treatment and the single most important step to help them recover is to '''quickly add media to the cells''' once they've been electroporated. The process is also very sensitive to salts in the DNA, and if you pipet too much DNA on to the cells, the extra salt may cause an electrical "arc," (you'll know this has happened from the flash of light and the "pop" you'll hear) frying the cells dead. If this happens, please get another aliquot of cells from the faculty and try the electroporation again, with less DNA. |
#When you are ready to electroporate the library, retrieve an aliquot of cells from the teaching faculty, a sterile cuvette, and an aliquot of rich, pre-warmed "SOC" media. | #When you are ready to electroporate the library, retrieve an aliquot of cells from the teaching faculty, a sterile cuvette, and an aliquot of rich, pre-warmed "SOC" media. | ||
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#Pipet 2 ul of the library DNA that is being held in an icebucket on the teacher's bench into your aliquot of cells. Be sure to decide and then note if you are screening the K+ or the P+ library. | #Pipet 2 ul of the library DNA that is being held in an icebucket on the teacher's bench into your aliquot of cells. Be sure to decide and then note if you are screening the K+ or the P+ library. | ||
#Let the cells and the DNA incubate on ice one minute. | #Let the cells and the DNA incubate on ice one minute. | ||
− | #Transfer 50 ul of the cells to the chilled cuvette and recover it with the blue lid. | + | #Transfer 50 ul of the cells (or more if the tube has more volume) to the chilled cuvette and recover it with the blue lid. |
#Put on your safety goggles. | #Put on your safety goggles. | ||
#Tap the cuvette on the bench so the cells rest in the bottom of the cuvette. | #Tap the cuvette on the bench so the cells rest in the bottom of the cuvette. | ||
− | #With the cuvette's "nub" facing | + | #With the cuvette's "nub" facing away from you, slide the cuvette into the electroporation chamber. Push the slide into the chamber until the cuvette is between the metal contacts. The lid on the cuvette will seem to block the path but in fact, it doesn't block the slider if you've lined thing up. |
− | + | #Make sure the electroporator is set to "Ec2" | |
− | + | ||
#Hold the pulse button until you hear a beep. | #Hold the pulse button until you hear a beep. | ||
− | #'''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 | + | #'''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 | + | # 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 and 3 of today's protocols. |
− | # Spread | + | # Spread 10 ul + 50 ul sterile water on one LB+Cam+Amp petri dishes. Plate 50 ul of the electroporation mix on another LB+Cam+Amp 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 or the dark (depending on the mutant you're looking for) until next time. |
===Part 2: Registry of Standard Biological Parts=== | ===Part 2: Registry of Standard Biological Parts=== | ||
− | What would it take to make DNA serve as a low-level programming language so that a genome is simply a particular program? DNA, like software, has an alphabet but with only four letters in the genetic code. Since there are proof-reading mechanisms in the "hardware," i.e. in the cell, syntax errors may be less likely to arise than in Python or Perl or C++. The code for cellular programs is messy but, honestly, so are computer programs. Subroutines are often dependent on one another (the cell cycle and DNA replication for example) and parts of the program get reused in useful, but complicated and unpredictable ways (seen as cross-talk in signaling pathways for example). Genetic code and computer code are both susceptible to viruses that highjack normally benign functions. 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 natural events | + | What would it take to make DNA serve as a low-level programming language so that a genome is simply a particular program? |
+ | *DNA, like software, has an alphabet but with only four letters in the genetic code. | ||
+ | *Since there are proof-reading mechanisms in the "hardware," i.e. in the cell, syntax errors may be less likely to arise than in Python or Perl or C++. | ||
+ | *The code for cellular programs is messy but, honestly, so are computer programs. Subroutines are often dependent on one another (the cell cycle and DNA replication for example) and parts of the program get reused in useful, but complicated and unpredictable ways (seen as cross-talk in signaling pathways for example). | ||
+ | *Genetic code and computer code are both susceptible to viruses that highjack normally benign functions. | ||
+ | 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 |