20.109(F07): M13.1
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.
Over the last few weeks you have gained important and detailed understanding of the natural M13 bacteriophage. Now you will help design a surrogate M13. A rough sketch of a more modular genome is included in today's lab. Today you and your lab partner should examine the draft and refine it. Based on everyone's design ideas, we will compile the surrogate M13 to test later in the term.
Part 1: M13.1
This is a rough sketch to refine before we request a DNA synthesis company to compile the program for us. All elements are specified at the Registry of Standard Biological Parts. Proposed refinements can be noted there 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: 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.
