Daniel Kluesing

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sub cloning: [[1]] sub cloning and ligation: [[2]]

APE Sequence annotation
1) Start APE from the shortcut
2) Load the dna file of interest from file->open
3) Clear the existing annotations by pressing ctrl-shift-k at the same time or by going to the ‘feature’ menu and selecting clear features
4) Select all bases by pressing ctrl-a
5) Annotate all features by pressing ctrl-k
6) Save the file

Adding a sequence to the feature library
1) from the feature menu select ‘edit feature library’
2) From the library screen click ‘new’
3) Enter the name of the feature
4) Enter the sequence of the feature
5) Select a color for forward and reverse (should be the same, press ‘same’ button to make this happen
6) Repeat for each new feature, when done, click save
7) Each dna file needs to be re-annotated by clearing and re-labeling

Riboregulator paper [[http://www.nature.com/nbt/journal/v22/n7/full/nbt986.html ]]
DNAEngine PCR data sheets [[3]]

Name of annotation software chris showed?
idiots guide to F-plasmid: [[4]]
RP4: [[5]]
TraJ = Relaxosome => starts dna transfer by making nick [[6]]
What is a DNA Nick? => in our case, a hole in the phosphate bond cause by an enzyme

2005 project
Why did it not work?
'Fluorescence is sometimes inhibited after conjugation' - is it known why?
'Slight leakiness of the lock we designed' - is it known why?
'Prove that our biobricked OriT-F plasmids is mobilizable' - slide 28, how'd they test Fluorescence if they don't know if they can mobilize the plasmid?
How 'strong' can we make the abstraction layer between conjugation and riboregulation?
What are the distance restrictions between ribsome binding site and start codon? (Empirical or theoretical?)
Five base address space => Two lock sequences/two conjugation plasmids => two possible messages?
Or 5 base, 4 possible options, addressSpace = 5^4 theoretical max messages?
Each message requires a unique conjugation plasmid? (Rather, each lock?) Or can the same type of plasmid carry different locks? (I think yes because of the modular design? But, require plasmids that don't conjugate with each other, hence the selection of F and RP4?)
'Unforeseen Eco site site-mutagenesis time' on slide 26, what's that?
'Create a ‘stop’ message to end communication after the programs are received or a reset method to return the cells to their original state' - can we do 'meta-data'? ie, Fluorescence on for 5 minutes, then self turn off?

Kinda fluffy and random but on my mind
Are there any metrics on the fidelity of biobricked functions? (ie, limits on length?)
Is there a measure of 'complexity' for a biobricked functional unit? (PoPs is I/O rate?)
In general, are there quality metrics other than the Boolean end result? (I guess no)
Can you 'unit test' constructions?
How much can be simulated prior to going into the cell?

Obtain a copy of the Issac guy's paper on locking

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