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Introduction

The ONBY ncAA used for our photocaging project. When exposed to 365 nm light, the ONB group is released, resulting in a normal tyrosine amino acid.

Using non-canonical amino acid incorporation, we recreated a light-activatable T7 polymerase (RNAP) for the spatio-temporal control of protein expression. The light-activatable T7 RNAP was created by mutating a Tyrosine codon at position 639 of the O-helix (figure??? of helix?), which is a domain crucial for polymerization of RNA during transcription. For this project, Y639 was mutated to an amber stop codon and we used ortho-nitrobenzyl tyrosine (ONBY), which is a "photocaged" ncAA. Thus, if our synthetase/tRNA pair works, position 639 should contain ONBY in place of Y. This work is essentially a recapitulation of earlier work done by [reference authors/paper].

When the photocaged ONBY is incorporated at position 639, RNAP activity is halted. The polymerase can become activated only upon de-caging of the ONB group from the ONBY. This is accomplished by exposing the cells to 365 nm light. When exposed to 365 nm light, the ONB group is released, resulting in position 639 now containing a normal tyrosine amino acid. T7 RNAP was selected because of its orthogonal nature, which allows us to selectively induce the expression of specific genes that are preceded by the T7 RNAP promoter. Because T7 promoters are not natively found in E.Coli, a gene downstream of a T7 promoter may be exclusively expressed through the introduction of 365 nm light.

Background

Tyrosine residue 639 (Y639) was specifically targeted because it lies on a crucial position on the O-helix and has been proved to be essential for polymerization. (REFERENCE) The Y639 residue in the O-helix is responsible for two major roles. First, this tyrosine residue discriminates between deoxyribose and ribose substrates. Second, Y639 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, Y639 proved to be a good candidate for incorporating a photocaged amino acid (Reference; You are referring to a specific result).

Incorporation of ONBY at position 639 halts the activity of the T7 polymerase because of the bulky nature of ONBY (REFERENCE). 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 (accurate?). Once the ONB group is removed, a normal tyrosine residue is left which restores RNA polymerase activity.

In order to incorporate the ncAA into amberless E.coli (which is described [here]), a Methanocaldoccus jannaschii tyrosyl-tRNA synthetase/tRNA pair was previously 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 the library was selected for its ability to charge ONBY while discriminating against other canonical amino acids. The resulting mutant ONBY synthetase contained five mutations (reference). The Asp 158→Ser 158 and Tyr 32→Gly 32 mutations are believed to result in the loss of hydrogen bonds with the natural substrate, which would disfavor binding to tyrosine, while the Tyr 32→Gly 32 and Leu 65→Gly 65 mutations are believed to increase the size of the substrate-binding pocket to accommodate the bulky o-nitrobenzyl group. The fifth mutation is ?? ARE THESE MUTATIONS CORRECT? Y32G is listed twice, which is fine, but then we should list the other mutations and what their roles are or that their roles are unknown.

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

@@ Line 93: / Line 93: @@
 =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.
+Tyrosine residue 639 (Y639) was specifically targeted because it lies on a crucial position on the O-helix and has been proved to be essential for polymerization. (REFERENCE) The Y639 residue in the O-helix is responsible for two major roles. First, this tyrosine residue discriminates between deoxyribose and ribose substrates. Second, Y639 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, Y639 proved to be a good candidate for incorporating a photocaged amino acid (Reference; You are referring to a specific result).
-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.
+Incorporation of ONBY at position 639 halts the activity of the T7 polymerase because of the bulky nature of ONBY (REFERENCE).  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 (accurate?). 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.
+In order to incorporate the ncAA into amberless E.coli (which is described '''[here]'''), a  ''Methanocaldoccus jannaschii'' tyrosyl-tRNA synthetase/tRNA pair was previously 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 the library was selected for its ability to charge ONBY while discriminating against other canonical amino acids.  The resulting mutant ONBY synthetase contained five mutations (reference).  The Asp 158→Ser 158 and Tyr 32→Gly 32 mutations are believed to result in the loss of hydrogen bonds with the natural substrate, which would disfavor binding to tyrosine, while the Tyr 32→Gly 32 and Leu 65→Gly 65 mutations are believed to increase the size of the substrate-binding pocket to accommodate the bulky o-nitrobenzyl group.  The fifth mutation is ??  ARE THESE MUTATIONS CORRECT?  Y32G is listed twice, which is fine, but then we should list the other mutations and what their roles are or that their roles are unknown.

Team:Austin Texas/photocage

From 2014.igem.org