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Lecture 19

Biology 1002B Lecture Notes - Lecture 19: Chlamydomonas, Phototaxis, Lysis

Course Code
BIOL 1002B
Tom Haffie

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Optogenetics II
How to look at biological function:
Biochemical Approach:
Grind up -eyespot, gather proteins from there
Isolated the channelrhodopsin protein
Study the protein to look at biological function
Genetics Approach:
Use mutant (that could not respond to light) to see what codes for
Both are brought together in molecular biology
How easy is it to go from protein sequence to gene sequence?
Wants to study gene instead of protein
Really easy to sequence protein.
The Problem of Redundancy
Much harder to go from protein sequence to gene sequence
More than one codon can code for the same amino acid
Coding sequence for channelrhodopsin
DNA equivalent to the mRNA (codons need to make the functional protein)
No introns
STOP at the end of eYFP
2 genes are “fused” together, makes one protein (translated together)
It becomes a CHIMERIC protein due to attachment of eYFP
Both proteins are still functional!
Not always the case, most of the time sticking another protein on
interferes with the function of the original protein.
Do this so you can track ChR2
Use light to excite YFP (different wavelength from channelrhodopsin)
Where Do We Find These Sequences?
Repository or molecular information (DNA, proteins, genome)
Deposit and Search databases (GenBac…)
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Lots of sequences available (easier and cheaper to sequence genome)
The sequence is great, but we need the actual piece of DNA
Polymerase Chain Reaction (PCR)
Amplification of specific DNA (just want a small section of the entire genome)
Need at least ONE COPY of the template DNA
In theory, this can be a single piece of DNA
Need a polymerase that can add A,C,T, Gs to the primer
Taq DNA polymerase
Can add 1000 bases per min (dATP, dGTP, dCTP, dDTTP)
Optimal activity at 72
Isolated from thermophiles
heat stable
won’t denature as fast
don’t need to be continuously replenished
PCR is incredibly specific
Taq polymerase like all polemorease, need a free 3’ end to be
able to add bases
Thus you need 2 primers specific to the DNA (12-30 bases long)
Primers are super specific
1/65536 chance of finding a sequence that match 16
PCR Reaction cycle
■ Denaturation
Heat the DNA to break the hydrogen bond (95 )
2 single strands
■ Annealing
Stick a primer on each end (high in concentration, so that the 2
strands do not simply anneal back together)
Lower the heat to that new bases can added (45 -65 )℃ ℃
■ Extension
Taq polymerase extends the new strand starting from the 3’ primer
Repeat to get more DNA
Why is amplification important?
You can do something now
manipulated it, cut it, run analysis on it, sequence it, etc
Before, didn’t have enough DNA to do anything and too much noise (mixed in with other
PCR Example
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