Difference between revisions of "20.109(F07): M13.1"
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"Divide and conquer" may be an effective military strategy but its usefulness is not limited to that arena. The reductionist approach has been an important means of understanding complex biological processes. By tweezing apart networks and pathways, the components that contribute to the overall behavior of the system may be understood in great detail. As you've seen, however, reassembly of the component level understanding into a predictive and quantitative models for the system isn't always straightforward. That's what happened with the T7 model that you read about last time, and the author's response to the limited success of the models is what makes that T7 work so novel. Rather than continue to tweeze apart and better understand the natural example, they built a surrogate T7 that was a better template for experimental work, easier to manipulate and analyze, easier to characterize and understand. | "Divide and conquer" may be an effective military strategy but its usefulness is not limited to that arena. The reductionist approach has been an important means of understanding complex biological processes. By tweezing apart networks and pathways, the components that contribute to the overall behavior of the system may be understood in great detail. As you've seen, however, reassembly of the component level understanding into a predictive and quantitative models for the system isn't always straightforward. That's what happened with the T7 model that you read about last time, and the author's response to the limited success of the models is what makes that T7 work so novel. Rather than continue to tweeze apart and better understand the natural example, they built a surrogate T7 that was a better template for experimental work, easier to manipulate and analyze, easier to characterize and understand. | ||
− | + | Now you will help design a surrogate M13 as well as consider a synthetic host for the designed phage. | |
===Part 1: M13.1=== | ===Part 1: M13.1=== | ||
− | + | A rough sketch of a more modular M13 genome is included here. All elements are specified at the [http://parts.mit.edu/registry/index.php/Main_Page Registry of Standard Biological Parts]. Today you and your lab partner should examine the draft and refine it. Proposed refinements can be noted on the individual parts as well as on our [[20.109(F07):Module 1:RefactorM13| M13 refactoring workpage]] | |
{| border="1" | {| border="1" | ||
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<br> | <br> | ||
− | ===Part 2: Sequencing=== | + | ===Part 2: Minimal E. coli genomes=== |
+ | |||
+ | ===Part 3: Sequencing=== | ||
From your experiment last time, you may feel confident that your oligonucleotide pair was successfully inserted into the M13K07 genome. What's unclear, however is which direction the insert has gone in. The restriction sites are so close together it won't be possible to know by restriction analysis if the construct you have in hand is in the intended or the reverse direction. For this, it will be necessary to sequence the region. | From your experiment last time, you may feel confident that your oligonucleotide pair was successfully inserted into the M13K07 genome. What's unclear, however is which direction the insert has gone in. The restriction sites are so close together it won't be possible to know by restriction analysis if the construct you have in hand is in the intended or the reverse direction. For this, it will be necessary to sequence the region. | ||
+ | |||
+ | |||
+ | DONE! | ||
+ | |||
+ | ===For Next Time=== | ||
+ | |||
+ | ===Reagents=== |
Revision as of 10:27, 3 July 2007
Contents
Introduction
"Divide and conquer" may be an effective military strategy but its usefulness is not limited to that arena. The reductionist approach has been an important means of understanding complex biological processes. By tweezing apart networks and pathways, the components that contribute to the overall behavior of the system may be understood in great detail. As you've seen, however, reassembly of the component level understanding into a predictive and quantitative models for the system isn't always straightforward. That's what happened with the T7 model that you read about last time, and the author's response to the limited success of the models is what makes that T7 work so novel. Rather than continue to tweeze apart and better understand the natural example, they built a surrogate T7 that was a better template for experimental work, easier to manipulate and analyze, easier to characterize and understand.
Now you will help design a surrogate M13 as well as consider a synthetic host for the designed phage.
Part 1: M13.1
A rough sketch of a more modular M13 genome is included here. All elements are specified at the Registry of Standard Biological Parts. Today you and your lab partner should examine the draft and refine it. Proposed refinements can be noted on the individual parts as well as on our M13 refactoring workpage
genetic element | promoter | RBS | coding |
---|---|---|---|
start synthesis with | |||
gII | BBa_M13102 (need 5' UTR?) |
BBa_M13502 | BBa_M13002' (modified to remove gene 10 promoter) |
gX | BBa_M13110 (need 5' UTR?) |
BBa_M13510 | BBa_M13010' (modified to remove gene 5 promoter) |
gV | BBa_M13105 (need 5' UTR?) |
BBa_M13505 | BBa_M13005 |
gVII | BBa_M13507 | BBa_M13007' (modified to remove overlap with gene 9 dwnstm) | |
gIX | BBa_M13509 | BBa_M13009' (modified to remove overlap with gene 8 dwnstm) | |
gVIII | BBa_M13108 (need 5' UTR?) |
BBa_M13508 | BBa_M13008 |
Transcriptional terminator (if M13K07 part, then need to modify to remove gene 3 promoter) | |||
gIII | BBa_M13103 | BBa_M13503 | BBa_M13003' (modified to remove gene 6 promoter, change GTG start?) |
gVI | BBa_M13106 | BBa_M13506 | BBa_M13006' (modified to remove gene 1 promoter) |
gI | BBa_M13101 | BBa_M13501 | BBa_M13001' (modified to remove gene 11 RBS, gene 4 promoter, RBS, start) |
gXI | BBa_M13511 | BBa_M13011' (modified to remove gene 4 promoter, RBS, start) | |
gIV | BBa_M13104 (need 5' UTR?) |
BBa_M13504 | BBa_M13004' |
M13K07 ori/KanR/p15a ori | |||
end synthesis |
Note: modified parts codon varied to remove direct repeats.
Part 2: Minimal E. coli genomes
Part 3: Sequencing
From your experiment last time, you may feel confident that your oligonucleotide pair was successfully inserted into the M13K07 genome. What's unclear, however is which direction the insert has gone in. The restriction sites are so close together it won't be possible to know by restriction analysis if the construct you have in hand is in the intended or the reverse direction. For this, it will be necessary to sequence the region.
DONE!