Matt Gethers

I'm currently a sophomore in course 20. When I'm behaving myself, I'm allowed to work in the Endy Lab at MIT. I will be documenting my UROP projects here. I will put my 20.109 work directly below.

20.109 Work

M13 Engineering Ideas

Extractions, separations, etc. include ensuring that each gene has its own promoter, RBS, terminator, and any other pertinent regulatory sequences. In addition to separating the genes, it would also be wise to develop or utilize/modify an existing system like Biobricks to build the genome so that convenient restriction sites exist between the genes so they can be easily removed or replaced. I used the following annotation to locate and design changes to the genome.

Gene	Modification
X	Extract from gene II.
II	Extract gene X.
V	Add some base pairs between V and VII to allow for a restriction site.
VII	Separate from gene IX.
III	Change the GTG to ATG Start?
VI	Add some base pairs between III and VI and VI anda I to allow for restriction sites.
I	Separate from genes IV and XI.
XI	Separate from genes I and IV.
IV	Separate from genes I and XI.
M13 ORI 1 & 2	Are two ORIs necessary? If so, can they be consolidated?
KanR	Possible to remove bps 6600 - 7100 upstream of KanR? Does this DNA have functional significance?

We didn't determine the concentration of the prepped DNA from the ligation reactions, so we cannot calculate the transformation efficiency of these transformations. We do know, however, that 5 ng of vector was used to transform the control plasmid, so efficiency = (1184 colonies)/(5*10^-3 micrograms of DNA)= 5.92*10^6 colonies/microgram of DNA.

'	2.27.07 Ligation and Transformation Data	'
DNA Ligation Sample	Expected Number of Transformants	Observed Number of Transformants
Control Plasmid	Many	1184
BKB with cocktail but no ligase	0	0
BKB with ligase and cocktail	0	6
BKB with insert and ligase and cocktail #1	A few	0-1
BKB with insert and ligase and cocktail #2	A few	5