20.109(F07): M13.1

Introduction

image credit: Justin Lo, MIT Course 20

"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

Refactor the M13K07 genome from the unique HpaI site in gene II through the unique BamHI site in gene III, guided by the algorithm described in the Refactoring T7 paper Supplementary Figure 1. Start by annotating the M13K07 sequence across this region, giving hypothetical part names ("BBb_M13_109.07_00001") to all genetically encoded functions. Collect these parts in a table and write a brief component descriptions for each. If there are particular aspects of the M13K07 sequence that cannot be edited, note these difficulties on your parts table, describing the design impass in sufficient detail that someone unfamiliar with our work might understand enough to help.

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 3: Sequencing

The invention of automated sequencing machines has made sequence determination a fast and inexpensive endeavor. The method for sequencing DNA is not new but automation of the process is recent, developed in conjunction with the massive genome sequencing efforts of the 1990s. At the heart of sequencing reactions is chemistry worked out by Fred Sanger in the 1970’s which uses dideoxynucleotides.

Normal bases versus chain-terminating bases

These chain-terminating bases can be added to a growing chain of DNA but cannot be further extended. Performing four reactions, each with a different chain-terminating base, generates fragments of different lengths ending at G, A, T, or C. The fragments, once separated by size, reflect the DNA’s sequence. In the “old days” (all of 10 years ago!) radioactive material was incorporated into the elongating DNA fragments so they could be visualized on X-ray film (image on left). More recently fluorescent dyes, one color linked to each dideoxy-base, have been used instead. The four colored fragments can be passed through capillaries to a computer that can read the output and trace the color intensities detected (image on right). Your sample will be sequenced in this way on an ABI 3730 DNA Analyzer.

Sequencing gel

Sequence trace data

Sequencing will be one of the last steps to assess the success of your work on the gene for p3. From your experiment last time, you may feel confident that your oligonucleotide pair was successfully inserted into the M13K07 genome. What remains unclear, however is which direction the insert has gone in. Since the restriction sites are so close together, the orientations can't be distinguished by restriction analysis. To know if the construct you have in hand is in the intended or the reverse direction, it will be necessary to sequence the region.

Sequencing reactions require 200-500 ng of plasmid DNA, and 3.2 pmoles of sequencing primer in a final volume of 12 μl. The miniprep'd plasmid should have ~1 ug of nucleic acid/ul but that will be a mixture of RNA and DNA, so we will guess at the amount of plasmid DNA there and use 1 ul of your miniprep'd DNA/sequencing reaction.

Prepare an eppendorf tube with enough material for two sequencing reactions, namely

• 2 ul of your plasmid DNA candidate(s)
• 6.4 ul of a 1:100 dilution of the sequencing primer
• 15.6 ul sterile water

Pipet this solution up and down to mix and then transfer 12 ul to one of the tubes in the 8-tube strip we will turn in at the biopolymer's facility in E17 for sequencing. The data will be available for you to examine one week from today. Keep your fingers crossed.

DONE!

For Next Time

1. Exchange writing assignment with lab partner for comments/reactions.

Reagents list

• Sequencing primers
  • M13KO7seq_2101_fwd
    • NO198 5'- CAA GGC ACT GAC CCC GTT AAA ACT TAT TAC -3'
  • M13KO7seq_2151_fwd
    • NO199 5'- AAA AGC CAT GTA TGA CGC TTA CTG GAA CGG -3'