Photocage Project

Introduction

Using the method of non-canonical amino acid incorporation into Amber stop codons, the University of Texas iGEM team created a light-activatable T7 polymerase (RNAP) for the spatio-temporal control of protein expression. The light-activatable T7 RNAP was created by mutating an amber stop codon at position 639 of the O-helix, which is a crucial domain involved in the polymerization of RNA. Ortho-nitrobenzyl tyrosine (ONBY), which is a "photocaged" ncAA, was then incorporated at that position to halt the activity of RNAP. By inducing the polymerase with non-phototoxic UV light, the ONB group of the non-canonical amino acid is cleaved, allowing the RNAP to function properly. Because of its orthogonal nature, T7 RNAP will only induce the expression of proteins that are preceded by a T7 promoter. Because T7 promoters are not natively found in E.Coli, a plasmid containing a protein bound to a T7 promoter may be exclusively expressed through the introduction of 365 nm light.

Background

The tyrosine residue (Tyr639) 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.

In order to incorporate the non-canonical amino acid into the amberless E.coli (which is described [here]), a Methanococcus jannaschii synthetase/tRNA pair had to be mutated to selectively charge and incorporate ONBY. Six residues (Tyr 32, Leu 65, Phe 108, Gln 109, Asp 158, and Leu 162) on the original synthetase were randomized and selected for its ability to charge ONBY while discriminating against other canonical amino acids. The Asp 158→Ser 158 and Tyr 32→Gly 32 mutations were likely involved in the loss of hydrogen bonds with the natural substrate, which would disfavor binding to natural tyrosine. On the other hand, the Tyr 32→Gly 32 and Leu 65→Gly 65 mutations likely increase the size of the substrate-binding pocket to accommodate the bulky o-nitrobenzyl group.

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

Results