Amino acids not included in the native 20 are commonly known as non-canonical amino acids (ncAAs). These ncAAs have unique chemistry that can provide very useful functionality not normally present in an organism. For our purposes, ncAAs were used to prevent protein functionality until spatially and/or temporally triggered. This summer our team has been working on re-creating a light-activatable T7 RNA Polymerase (RNAP) for a method of non-invasive, spacio-temporal control of protein expression. T7 RNAP was our enzyme of choice for this project due to the presence of a tyrosine residue in the active site of the polymerase. By recoding this tyrosine residue, ortho-nitrobenzyl tyrosine was incorporated into the active site thus acting as a molecular cage. T7 RNAP is only functional when exposed to a certain wavelength of light that cleaves a molecular cage from the polymerase’s active site. In our experiments, ortho-nitrobenzyl tyrosine (ONBY) was used as our photocaged ncAA. ONBY was used because once the ONB group is cleaved off, the ncAA functions as a normal tyrosine. This proved to be particularly useful because T7 polymerase has a tyrosine residue in its active site that is necessary for proper function of the protein. Once de-caged, the polymerase is free to transcribe sequences that are preceded by a T7 promoter. GFP was used as a reporter to analyze and optimize each construct for spacio-temporal specificity. In addition, GFP was used to examine the effect of a certain non-canonical amino acid on fluorescence when placed in the fluorophore.
Background
The tyrosine residue which is replaced by ONBY lies on a crucial position on the O-helix and has been proved to be essential for polymerization. The Tyr639 residue in the O-helix is responsible for two major roles. First, this tyrosine residue discriminates between deoxyribose and ribose substrates. Second, Tyr639 is responsible for moving newly synthesized RNA out of the catalytic site and preparing for the next NTP to be inserted. These functions of the O-helix were shown to be essential through mutational analysis. Because the loss of this tyrosine residue in the active site leads to a non-functional polymerase, Tyr639 proved to be a good candidate for incorporating a photocaged amino acid.
Because of the bulky nature of ONBY, its incorporation at position 639 essentially halted the activity of the T7 polymerase. However, the bulky ONB group is able to be removed through irradiation with 365 nm light. The wavelength of light used to "decage" the amino acid proved to be another advantage because 365 nm light is not toxic to the cell. Once the ONB group is removed, a normal tyrosine residue is left which restores RNA polymerase activity.
Experimental Methods
Note: I will elaborate in more words in the following sections
Preparation & Growth of Cultures
Picked Colonies
PC 1: pQE/ONBY
PC 2: pQE/ONBY
PC 3: MJH117/ONBY
PC 4: MJH117/ONBY
PC 5: aT7/ONBY
PC 6: aT7/ONBY
Antibiotics for PC 1-4: Zeo, Kan, Gent
Antibiotics for PC 5-6: Zeo, Crb, Gent
Conditions for each picked colony
(-) IPTG, (-) ONBY
(+) IPTG, (-) ONBY
(+) IPTG, (+) ONBY
Inoculate each tube with designated PC; add 1 uL of cells into each tube
Grow cultures for 2 hours
Add IPTG to all (+) IPTG culture tubes
Grow another 2-4 hours
Decaging ortho-nitrobenzyl tyrosine
Take cultures out and irradiate with 365 nm light
Place all (+) IPTG (+) ONBY cells into wells of a 12-well plate
Place (+) IPTG (-) ONBY of PC 1, 3, 5 into wells
Place (-) IPTG (-) ONBY of PC 3 & 4 to use as a control for growth without ONBY (*this will allow us to see how much the ONBY solution decreases growth rate of cells)
Place negative control into 12 well plate
Irradiate using handheld blacklight placed directly over the wells; irradiate cells and take out at every time point (0 min, 1 min, 5 min, 15 min, 30 min) and transfer to wells in a 96 well plate to test fluorescence
Measuring Fluorescence
NEED TO EDIT: THIS WAS CUT AND PASTED FROM A PAPER WE WROTE.
BELOW: BETTER IMAGE resolution
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