Jessica Keenan: M13 Renovation

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Contents

Day 6 For Next Time

The p3 protein is about 48kDa, so we will expect to see a blot near (a bit below) the 50kDa blot on the standard (fifth blot from the top).

Day 5 For Next Time

The gel figure can be found here.

Day 4 For Next Time

Transformations

Plate Results Expected Transformation Efficiency
positive control plasmid 517 lots (517 colonies/5ng plasmid DNA)*(1000ng/ug) = 1.03E5 colonies/ug plasmid DNA
backbone -ligase none none 0 colonies/ug plasmid DNA
backbone + ligase none a few 0 colonies/ug plasmid DNA
backbone + ligase + insert none more 0 colonies/ug plasmid DNA

Diagnostic Digest 1

Colony Actual Band Size (bp) Expected Band Size (bp)
1 >10kb, ~7kb 5714, 2970
2 >10kb, ~7kb 5714, 2970
3 >10kb, ~7kb 5714, 2970
4 >10kb, ~7kb 5714, 2970

Diagnostic Digest 1 did not work as expected. This gel looks like the gel we ran previously with uncut plasmid: there is one band at about 7kb representing supercoiled plasmid, and another band at greater than 10kb representing non-supercoiled uncut plasmid. It appears that neither enzyme cut the plasmid, which could mean there was some problem with the reaction (for example, a reagent was not added properly). It is probably not just a problem with the insert, as the XhoI was cutting a part of the plasmid that was not on the insert - perhaps this digest should be performed again.

Diagnostic Digest 2

Colony Actual Band Size (bp) Expected Band Size (bp)
1 ~7kb, ~3kb, ~6kb 5810, 2874
2 ~7kb, ~3kb, ~6kb 5810, 2874
3 ~7kb, ~3kb, ~6kb 5810, 2874
4 ~7kb, ~3kb, ~6kb 5810, 2874

This digest appears to have largely worked as predicted. The bands at about 3kb and about 6kb represent the fragments cut by the restriction enzymes. The ~7kb band could be from circular (supercoiled) plasmid that was not digested (though there is no band for uncut non-supercoiled plasmid). It seems that at least the FspI site (and also the natural MscI site) works properly.


Day 3 For Next Time

Problems 1, 2, and 4 may be found here. (Problems 1 and 4 were done with equation editor - if they don't come up, click on the blank area where they should be a couple times)

Diagnostic digest 1 plasmid with insert plasmid no insert
Enzyme(s) used BglII XhoI
Buffer used Buffer 3 Buffer 3
Temperature 37C 37C
Cut Site 5' A^GATC_T 5' C^TCGA_G
Predicted fragments 5718 bp, 2968 bp 8669 bp
Diagnostic digest 2
Enzyme(s) used SfoI HindIII
Buffer used Buffer 2 Buffer 2
Temperature 37C 37C
Cut Site 5' GGC^GCC 5' A^AGCT_T
Predicted fragments 5188 bp, 3498 bp 8669 bp

Day 2 For Next Time

Gel Electrophoresis Data

Data from the gel run on the cut M13K07 backbone (9/13/07)

Titering

10ul x (1ml/10^3ul) x 10^12 PFU/ml x 10^-8 = 100 PFU

The D5 strain appears to be F-. Since the pilus (which requires an F plasmid) is required for the M13 to attach to the cell, there should not be any plaques.

Response: "synthetic biology is about engineering while genetic engineering is about biology"

While the statement "synthetic biology is about engineering while genetic engineering is about biology" is valid to a certain extent, it is an oversimplification that neglects the importance of biology to synthetic biology and the use of engineering concepts in genetic engineering. Synthetic biology is certainly a field that is largely based on engineering; it involves designing basic parts and using those parts to design and build larger biological systems. Biology, however, is also an integral part of this design process. Without thorough biological understanding of cellular systems, it would be nearly impossible to assemble biological parts to form a device with a predictable and reliable function. Moreover, as its name implies, genetic engineering relies on engineering concepts as well as biology. Biology is certainly important for determining which parts should be added to a system, and how they should be added, but one must also consider how adding these parts will affect the design of the genome. Thus, both biology and engineering are important aspects of genetic engineering and synthetic biology.

Day 1 For Next Time

Gene Gene Product Function Re-engineering Thoughts
I phage assembly
II dsDNA replication
  • separate from gene X
  • add a promoter to decrease the amount of + strand synthesized and possibly decrease number of phages produced
III binds to bacterial pilus (5 copies)
  • alter this product so that it is able to bind to a different receptor. The phage could then use other cell types as hosts
  • if a strong promoter is placed in front of this gene, would the increased amount of III facilitate binding to the host?
IV phage assembly separate from gene XI
V stabilizes ssDNA helix flourescently lab V and VIII (so that they are sensitive to different wavelengths) to visualize the the transfer of the ssDNA from a 'coat' of V inside the cell, to a coat of VIII after it leaves the cell.
VI phage tail protein (5 copies)
VII phage head protein (5 copies) separate from gene IX
VIII phage coat protein (2700 copies) separate from gene IX
IX phage head protein (5 copies)
  • separate from gene VII
  • separate from gene VIII
X DNA replication separate from gene II
XI assembly separate from gene IV

M13's Relatives

M13 is closely related to the fl and fd phages, all of which belong the the bacteriophage family Inoviridae and have ssDNA. Their genomes are largely conserved, but there are some differences between their protein coats, which results in M13 having a lower surface charge.

BBa_M1307 as a Standard Part

This criticism might be made because BBa_M1307 represents the entire M13 genome (plus some resistance genes). Because the genome codes for several functions (replication, phage coat production, etc) it is arguably not a single biological 'part.' However, it seems that the M13 genome provides a sound and readily manipulated backbone for the addition of other parts (for example, a kanamycin resistance 'part' has already been added to it), and is therefore a useful tool to have in the library of standard parts.

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