Bacteriophage engineering methods

Patent metadata

Field	Value
Publication number	US-10815535-B2
Application number	US-201715470685-A
Country	US
Kind code	B2
Filing date	Mar 27, 2017
Priority date	Mar 28, 2016
Publication date	Oct 27, 2020
Grant date	Oct 27, 2020

Abstract

The present disclosure provides methods and kits for generating recombinant bacteriophage genomes.

First claim

The invention claimed is:

1. A method for generating a recombinant bacteriophage genome comprising:
(a) contacting a first bacteriophage genome comprising a unique first recognition site with a first restriction enzyme in vitro under conditions where the first restriction enzyme cleaves the first recognition site to produce a cleaved first bacteriophage genome; and
(b) recombining in vitro the cleaved first bacteriophage genome with a heterologous nucleic acid in the presence of a recombination system under conditions to produce a recombinant bacteriophage genome,
wherein the cleaved first bacteriophage genome comprises a first cleaved bacteriophage genomic fragment and a second cleaved bacteriophage genomic fragment,
wherein the heterologous nucleic acid comprises a 5′ flanking region that is homologous to the 3′ end of the first cleaved bacteriophage genomic fragment, and a 3′ flanking region that is homologous to the 5′ end of the second cleaved bacteriophage genomic fragment, and
wherein the homologous 5 ‘ flanking region of the heterologous nucleic acid, and the homologous 3’ flanking region of the heterologous nucleic acid, each has a length of 20-70 base pairs (bps);
wherein the heterologous nucleic acid encodes a non-endogenous protein; and
wherein the recombinant bacteriophage genome is capable of producing non-endogenous protein that is functionally active when transformed into a bacterial host cell.

2. The method of claim 1 , wherein the first bacteriophage genome corresponds to E. coli T7.

3. The method of claim 1 , further comprising propagating the recombinant bacteriophage genome in a bacterial host cell.

4. The method of claim 3 , wherein the bacterial host cell is a natural host of a bacteriophage comprising the first bacteriophage genome.

5. The method of claim 1 , wherein the first restriction enzyme is SwaI.

6. The method of claim 1 , wherein the first restriction enzyme is NheI.

7. The method of claim 1 , wherein the heterologous nucleic acid is about 500-1050 base pairs in length.

8. The method of claim 1 , wherein the recombination system comprises a 5′-3′ exonuclease, a DNA polymerase, and a DNA ligase.

9. The method of claim 1 , wherein the heterologous nucleic acid comprises an open reading frame that encodes a bioluminescent protein, a fluorescent protein, a chemiluminescent protein, or any combination thereof.

10. The method of claim 9 , wherein the open reading frame of the heterologous nucleic acid is operably linked to an expression control sequence that is capable of directing expression of the bioluminescent protein, the fluorescent protein, the chemiluminescent protein, or any combination thereof.

11. The method of claim 9 , wherein the bioluminescent protein is Aequorin, firefly luciferase, Renilla luciferase, red luciferase, luxAB, or nanoluciferase.

12. The method of claim 9 , wherein the chemiluminescent protein is β-galactosidase, horseradish peroxidase (HRP), or alkaline phosphatase.

13. The method of claim 9 , wherein the fluorescent protein is TagBFP, Azurite, EBFP2, mKalamal, Sirius, Sapphire, T-Sapphire, ECFP, Cerulean, SCFP3A, mTurquoise, monomeric Midoriishi-Cyan, TagCFP, mTFP1, EGFP, Emerald, Superfolder GFP, Monomeric Azami Green, TagGFP2, mUKG, mWasabi, EYFP, Citrine, Venus, SYFP2, TagYFP, Monomeric Kusabira-Orange, mKOκ, mKO2, mOrange, mOrange2, mRaspberry, mCherry, dsRed, mStrawberry, mTangerine, tdTomato, TagRFP, TagRFP-T, mApple, mRuby, mPlum, HcRed-Tandem, mKate2, mNeptune, NirFP, TagRFP657, IFP1.4, iRFP, mKeima Red, LSS-mKate1, LSS-mKate2, PA-GFP, PAmCherryl, PATagRFP, Kaede (green), Kaede (red), KikGR1 (green), KikGR1 (red), PS-CFP2, PS-CFP2, mEos2 (green), mEos2 (red), PSmOrange, or Dronpa.

14. The method of claim 10 , wherein the expression control sequence is an inducible promoter or a constitutive promoter.

15. The method of claim 1 , wherein the nucleic acid sequence of the recombinant bacteriophage genome comprises SEQ ID NO: 2.

16. The method of claim 1 , wherein the nucleic acid sequence of the recombinant bacteriophage genome comprises SEQ ID NO: 1.

17. The method of claim 1 , wherein the nucleic acid sequence of the recombinant bacteriophage genome comprises one or more of SEQ ID NO: 6 or SEQ ID NO: 7.

Assignees

• Charles Stark Draper Laboratory Inc

Inventors

• Holder Jason
• Mcbrine Connor
• Gruszka Sarah
• Rogers Miles
• Billings Nicole

Classifications

• C12N15/86 Primary

  Viral vectors · CPC title

• C12N2310/20

  involving clustered regularly interspaced short palindromic repeats [CRISPR] · CPC title

• B01L3/502753

  characterised by bulk separation arrangements on lab-on-a-chip devices, e.g. for filtration or centrifugation · CPC title

• B01L2300/12

  Specific details about materials · CPC title

• C12N7/00

  Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof (preparing medicinal viral antigen or antibody compositions, e.g. virus vaccines, A61K39/00) · CPC title