A Protocol for Functional Assessment of Whole-Protein Saturation Mutagenesis Libraries Utilizing High-Throughput Sequencing

Summary

We present a protocol for the functional assessment of comprehensive single-site saturation mutagenesis libraries of proteins utilizing high-throughput sequencing. Importantly, this approach uses orthogonal primer pairs to multiplex library construction and sequencing. Representative results using TEM-1 β-lactamase selected at a clinically relevant dosage of ampicillin are provided.

Abstract

Site-directed mutagenesis has long been used as a method to interrogate protein structure, function and evolution. Recent advances in massively-parallel sequencing technology have opened up the possibility of assessing the functional or fitness effects of large numbers of mutations simultaneously. Here, we present a protocol for experimentally determining the effects of all possible single amino acid mutations in a protein of interest utilizing high-throughput sequencing technology, using the 263 amino acid antibiotic resistance enzyme TEM-1 β-lactamase as an example. In this approach, a whole-protein saturation mutagenesis library is constructed by site-directed mutagenic PCR, randomizing each position individually to all possible amino acids. The library is then transformed into bacteria, and selected for the ability to confer resistance to β-lactam antibiotics. The fitness effect of each mutation is then determined by deep sequencing of the library before and after selection. Importantly, this protocol introduces methods which maximize sequencing read depth and permit the simultaneous selection of the entire mutation library, by mixing adjacent positions into groups of length accommodated by high-throughput sequencing read length and utilizing orthogonal primers to barcode each group. Representative results using this protocol are provided by assessing the fitness effects of all single amino acid mutations in TEM-1 at a clinically relevant dosage of ampicillin. The method should be easily extendable to other proteins for which a high-throughput selection assay is in place.

Introduction

Mutagenesis has long been employed in the laboratory to study the properties of biological systems and their evolution, and to produce mutant proteins or organisms with enhanced or novel functions. While early approaches relied on methods which produce random mutations in organisms, the advent of recombinant DNA technology enabled researchers to introduce select changes to DNA in a site-specific manner, i.e., site-directed mutagenesis^1,2. With current techniques, typically using mutagenic oligonucleotides in a polymerase chain reaction (PCR), it is relatively facile to create and assess small numbers of mutations (e.g., point mutations) in....

Protocol

Note: See Figure 1 for outline of protocol. Several steps and reagents in the protocol require safety measures (indicated with "CAUTION"). Consult material safety data sheets before use. All protocol steps are performed at RT unless other indicated.

1. Prepare Culture Media and Plates

1. Prepare and sterilize by autoclaving 1 L purified water, 100 ml Super Optimal Broth (SOB; Table 1), 1 L Luria-Bertani broth (LB; Table 2) and 1 L LB-agar (Table 3). Prepare separately and sterilize three culture flasks each containing 1 L LB.
   Note: Throughout the protocol "wat....

Results

The plasmid map for the five modified pBR322 plasmids containing orthogonal priming sites (pBR322_OP1 - pBR322_OP5) is shown in Figure 2A. To test whether the orthogonal primers are specific, PCRs were performed using each pair of orthogonal primers individually, along with all five pBR322_OP1-5 plasmids, or with all plasmids minus the plasmid matching the orthogonal primer pair. The correct product was only obtained when the matching plasmid was included, and no product .......

Discussion

Here a protocol is described for performing the functional assessment of whole-protein saturation mutagenesis libraries, using high-throughput sequencing technology. An important aspect of the method is the use of orthogonal primers during the cloning process. Briefly, each amino acid position is randomized by mutagenic PCR, and mixed together into groups of positions whose combined sequence length is accommodated by high-throughput sequencing. These groups are cloned into plasmid vectors containing pairs of orthogonal p.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Typtone	Research Products Intl. Corp.	T60060-1000.0
Yeast extract	Research Products Intl. Corp.	Y20020-500.0
Sodium chloride	Fisher Scientific	BP358-212
Potassium chloride	Sigma-Aldrich	P9333-500G
Magnesium sulfate	Sigma-Aldrich	M7506-500G
Agar	Fisher Scientific	BP1423-500
Tetracycline hydrochloride	Sigma-Aldrich	T7660-5G
Petri plates	Corning	351029
MATLAB	Mathworks	http://www.mathworks.com/products/matlab/
Oligonucleotide primers	Integrated DNA Technologies	https://www.idtdna.com/pages/products/dna-rna/custom-dna-oligos	25 nmol scale, standard desalting
pBR322_AvrII	available upon request		pBR322 plasmid modified to contain AvrII restriction site downstream of the TEM-1 gene
pBR322_OP1 – pBR322_OP5	available upon request		five modified pBR322 plasmids each containing a pair of orthogonal priming sites
Q5 high-fidelity DNA polymerase	New England Biolabs	M0491L	includes 5x PCR buffer and PCR additive (GC enhancer)
15 ml conical tube	Corning	430025
Multichannel pipettes (Eppendorf ResearchPlus)	Eppendorf
PCR plate, 96 well	Fisher Scientific	14230232
96 well plate seal	Excel Scientific	F-96-100
Veriti 96-well thermal cycler	Applied Biosystems	4375786
6x gel loading dye	New England Biolabs	B7024S
Agarose	Research Products Intl. Corp.	20090-500.0
Ethidium bromide	Bio-Rad	161-0433
UV transilluminator (FOTO/Analyst ImageTech)	Fotodyne Inc.	http://www.fotodyne.com/content/ImageTech_gel_documentation
EB buffer	Qiagen	19086
96-well black-walled, clear bottom assay plates	Corning	3651
Lambda phage DNA	New England Biolabs	N3011S
PicoGreen dsDNA reagent	Invitrogen	P7581	dsDNA quantitation reagent, used in protocol step 2.2.4
Victor 3 V microplate reader	PerkinElmer
DNA purification kit	Zymo Research	D4003
Microcentrifuge tubes	Corning	3621
Long-wavelength UV illuminator	Fisher Scientific	FBUVLS-80
Agarose gel DNA extraction buffer	Zymo Research	D4001-1-100
AatII	New England Biolabs	R0117S
AvrII	New England Biolabs	R0174L
T4 DNA ligase	New England Biolabs	M0202S
EVB100 electrocompetent E. coli	Avidity	EVB100
Electroporator (E. coli Pulser)	Bio-Rad	1652102
Electroporation cuvettes	Bio-Rad	165-2089
Spectrophotometer (Ultrospec 3100 pro)	Amersham Biosciences	80211237
50 ml conical tubes	Corning	430828
Plasmid purification kit	Macherey-Nagel	740588.25
8 well PCR strip tubes	Axygen	321-10-551
Qubit dsDNA HS assay kit	Invitrogen	Q32854	dsDNA quantitation reagent
Qubit assay tubes	Invitrogen	Q32856
Qubit fluorometer	Invitrogen	Q32866
Ampicillin sodium salt	Akron Biotechnology	50824296
MiSeq reagent kit v2 (500 cycles)	Illumina	MS-102-2003
MiSeq desktop sequencer	Illumina	http://www.illumina.com/systems/miseq.html	alternatively, one could sequence on Illumina HiSeq platform
FLASh software	John Hopkins University - open source	http://ccb.jhu.edu/software/FLASH/	software to merge paired-end reads from next-generation sequencing data
AatII_F			GATAATAATGGTTTCTTAGACG TCAGGTGGC
AvrII_R			CTTCACCTAGGTCCTTTTAAAT TAAAAATGAAG
AvrII_F			CTTCATTTTTAATTTAAAAGGA CCTAGGTGAAG
AatII_OP1_R			ACCTGACGTCCGTATTTCAAC TGTCCGGTCTAAGAAACCATT ATTATCATGACATTAAC
AatII_OP2_R			ACCTGACGTCCGCTCACGGA GTGTACTAATTAAGAAACCATT ATTATCATGACATTAAC
AatII_OP3_R			ACCTGACGTCGTACGTCTGA ACTTGGGACTTAAGAAACCA TTATTATCATGACATTAAC
AatII_OP4_R			ACCTGACGTCCCGTTCTCGAT ACCAAGTGATAAGAAACCATT ATTATCATGACATTAAC
AatII_OP5_R			ACCTGACGTCGTCCGTCGGA GTAACAATCTTAAGAAACCAT TATTATCATGACATTAAC
OP1_F			GACCGGACAGTTGAAATACG
OP1_R			CGACGTACAGGACAATTTCC
OP2_F			ATTAGTACACTCCGTGAGCG
OP2_R			AGTATTAGGCGTCAAGGTCC
OP3_F			AGTCCCAAGTTCAGACGTAC
OP3_R			GAAAAGTCCCAATGAGTGCC
OP4_F			TCACTTGGTATCGAGAACGG
OP4_R			TATCACGGAAGGACTCAACG
OP5_F			AGATTGTTACTCCGACGGAC
OP5_R			TATAACAGGCTGCTGAGACC
Group1_F			ACACTCTTTCCCTACACGAC GCTCTTCCGATCTNNNNNGC ATTTTGCCTACCGGTTTTTGC
Group1_R			GTGACTGGAGTTCAGACGTG TGCTCTTCCGATCTNNNNNTC TTGCCCGGCGTCAAC
Group2_F			ACACTCTTTCCCTACACGAC GCTCTTCCGATCTNNNNNGA ACGTTTTCCAATGATGAGCAC
Group2_R			GTGACTGGAGTTCAGACGTG TGCTCTTCCGATCTNNNNNGT CCTCCGATCGTTGTCAGAAG
Group3_F			ACACTCTTTCCCTACACGAC GCTCTTCCGATCTNNNNNAG TAAGAGAATTATGCAGTGCTGCC
Group3_R			GTGACTGGAGTTCAGACGTG TGCTCTTCCGATCTNNNNNTC GCCAGTTAATAGTTTGCGC
Group4_F			ACACTCTTTCCCTACACGAC GCTCTTCCGATCTNNNNNCC AAACGACGAGCGTGACAC
Group4_R			GTGACTGGAGTTCAGACGTG TGCTCTTCCGATCTNNNNNGC AATGATACCGCGAGACCC
Group5_F			ACACTCTTTCCCTACACGAC GCTCTTCCGATCTNNNNNCG GCTGGCTGGTTTATTGC
Group5_R			GTGACTGGAGTTCAGACGTG TGCTCTTCCGATCTNNNNNTAT ATGAGTAAACTTGGTCTGACAG
501_F			AATGATACGGCGACCACCGA GATCTACACTATAGCCTACAC TCTTTCCCTACACGAC
502_F			AATGATACGGCGACCACCGA GATCTACACATAGAGGCACA CTCTTTCCCTACACGAC
503_F			AATGATACGGCGACCACCGA GATCTACACCCTATCCTACAC TCTTTCCCTACACGAC
504_F			AATGATACGGCGACCACCGA GATCTACACGGCTCTGAACA CTCTTTCCCTACACGAC
505_F			AATGATACGGCGACCACCGA GATCTACACAGGCGAAGACA CTCTTTCCCTACACGAC
701_R			CAAGCAGAAGACGGCATAC GAGATCGAGTAATGTGACTG GAGTTCAGACGTG
702_R			CAAGCAGAAGACGGCATAC GAGATTCTCCGGAGTGACTG GAGTTCAGACGTG