site saturation scanning for every lab: supercharging site ... saturation... · scanning for every...

Site Saturation

Scanning for Every Lab:

Supercharging Site-

Directed Mutagenesis

Holly Hogrefe, Ph.D. R&D Manager

Genomics Solutions Division

Agilent Technologies

11/19/2014 1

11/19/2014

2

New Product Introduction:

QuikChange HT Protein Engineering System

• High-quality libraries, lacking the biases and unwanted mutations

associated with other mutagenesis techniques

• Custom oligonucleotide libraries used to achieve:

• An expanded menu of mutagenesis strategies, not

possible with standard or degenerate oligos:

• Create rationally-designed complex mutant libraries targeting:

• Entire proteins, up to 1000 codons per kit

• Alanine and site-saturation scanning

• Rational combinatorial mutagenesis

Definitions

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11/19/2014

Random mutagenesis: • Tool for identifying nucleotide or amino acid changes that alter function

• Error-prone PCR conditions used to introduce single bp changes at random

locations in a gene or promoter of interest

• Strategy most commonly used when the location of beneficial changes is unknown

• Advantages over rational (non-random) approaches:

• No prior knowledge of structure required

• Survey large mutational target regions, up to several kbp in size

Agilent Product: GeneMorph Random Mutagenesis kit

• Limitations:

• Screen large libraries to identify useful mutations

• Changes limited to single base substitutions; a large fraction of protein

sequence space is not accessible since most amino acid changes require

2-3 point mutations in the same codon

“With single base mutations, only 5.7 amino acid substitutions on average are accessible from any given

amino acid residue” (Miyazaki and Arnold (99) J Mol Evol 49:716–720)

11/19/2014

• Limitations:

• Throughput; 1 mutant per reaction

• Structural information is lacking in many cases

Agilent Products: QuikChange Site-Directed Mutagenesis kits

Site-Directed Mutagenesis: • Process of creating one or more nucleotide or codon changes in a gene or

regulatory sequence

• Rational (non-random) approach that employs DNA polymerase activity to

incorporate mutagenic oligos into wild-type DNA sequences

• Strategy used when location of desired modifications is known; potential target

sites can be identified by analyzing protein structures, amino acid sequence

homologies, or prior mutagenesis data

• Used to determine structure-function relationships in proteins or promoters, identify

post-translational modifications, define protein-protein interactions, modify enzyme

activity, optimize expression, add restriction sites, and correct unwanted mutations

The QuikChange Method

• Features a simple 3-step

protocol

• Creates single base changes,

codon replacements, insertions

and deletions with >80%

mutation efficiency

• Employs high-fidelity Pfu

polymerase and linear

amplification to avoid

unintended mutations

• Referenced in tens of

thousands of publications

MIX Denature plasmid and annealing

primers containing desired

mutation X

Gene in plasmid

with mutation

target site

Mutagenic

primers

CYCLE Temperature cycle to extend and

incorporate mutation primers

resulting in nicked circular strands

DIGEST Digest parental DNA template

TRANSFORM Transform the resulting

annealed double-stranded

nicked DNA molecules

After transformation,

XL-1Blue E. coli cell repairs

nicks in plasmid

11/19/2014

Definitions cont’d

11/19/2014

Agilent Product: QuikChange Multi Site-Directed Mutagenesis kit*

No. sequences required

No. sites to saturate: 1 2 3 4

Rational design 20 400 8000 160,000

Degenerate codons 64 4096 262,144 16,777,216

Scalability limited by transformation efficiency of E. coli and quality of degenerate codon libraries, as codon

redundancy and amino acid bias increase exponentially as the number of mutation sites is increased;

Site-saturation mutagenesis: • Process of determining the impact of every amino acid replacement at a single codon

• Most practical method uses degenerate oligos (referred to as “codon-saturation”);

oligos synthesized with an NNN codon comprise 64 unique sequences encoding 20

different amino acids at one position

• Site-saturation libraries can be screened to identify the best side chain replacements,

and required structural features (size, charge, etc.) for optimal activity

• Site-saturation with degenerate codons is less-than-ideal: • Bias toward amino acid changes encoded by redundant codons

• Frequency of unwanted termination and rare codons

• Limited scalability or target size:

No. oligos & rxns

4

160,000

1

N = nt position synthesized with equimolar

mixture of 25% AGCT)

Example of Site-Saturation Mutagenesis

11/19/2014

McElhinny et al (09) in Molecular Themes in

DNA Replication (Cox L.S., ed) Hogrefe (10) in In Vitro Mutagenesis Protocols (Bramam, J. ed) pg. 271-284

JDF-3 P410L DNA polymerase

(CCT→CTT)

Yield from random selection of 30 QuikChange mutants:

pJDF3

13%

Rare in

EPPCR library

(2 nt changes)

Random mutagenesis: Random mutant

library of Thermococcus sp. JDF-3 Pol B

was expressed in E. coli and screened for

radioactive-ddNTP uptake

Arezi et al (02) J. Mol. Biol. 322:719-729


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Alanine (or single mutation) scanning: • Systematic process for evaluating the functionality of a group of amino acids

• A collection of site-directed mutants is constructed, where each mutant

contains an alanine (or other neutral amino acid) substitution at a unique

location; after measuring impact to function, the information is used collectively

to map essential positions

Alanine scanning of the

HIV gp41 loop Jacobs et

al (05) JBC 280:27284-88

30 gp41 alanine mutants

were constructed using

the QuikChange kit with

30 primer pairs; viral entry

was measured using a

Luciferase reporter assay

100%

10%

0%

• Limitations: While powerful, alanine scanning is generally limited to peptides

or small domains due to the cost and amount of labor needed to construct

multiple single-site mutants (one QC reaction and primer pair per codon)


11/19/2014

Site-saturation scanning: • Combination of site-saturation and scanning mutagenesis strategies

Agilent Product: QuikChange HT Protein Engineering System

Hypothetical sequence-function map for a 25 amino acid

portion of a protein. The color of each square in the heat

map illustrates the functional consequence of individual

side chain substitutions (single-letter abbreviation) at each

position.

Deep mutational scanning: a new style of protein

science. Fowler & Fields (2014) Nature Methods

11:801-7

“By enabling the impact of mutations to be examined in

an unbiased fashion, deep mutational scanning can

reveal the unexpected”.

• When feasible, provides comprehensive structure-function analysis by surveying

the functional consequences of every side-chain replacement at each location

Significant limitations: Requires a high-throughput cost-effective method for

constructing site-directed mutants, in addition to substantial screening and sequencing

QuikChange HT

Protein Engineering

System

11/19/2014 11

12

+

QuikChange Lightning

Site-Directed Mutagenesis

Agilent’s SurePrint

Technology

Game-Changing Mutagenesis Technology

Quick, accurate, and cost-effective method for constructing

complex rationally-designed mutant protein libraries


11/19/2014


Rationally-designed oligo libraries provide:

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Constructing oligo libraries with QuikChange offers:

• Fast and easy one-day workflow for in-house library construction

• High incorporation efficiency with minimal polymerase-induced mutations

• Ultra-high transformation efficiencies for maximum library size

• Better quality libraries, more information content

• User-specified codon usage for optimal expression

• No unwanted codons; avoid biases associated with degenerate codons

• Reduced screening requirement

• Comprehensive custom content

• Up to 120,000 user-specified sequences

• Easy-to-use oligo library design tool

• Flexible design options, one or more proteins

• An expanded menu of mutagenesis strategies, not available using standard oligos

• Single-site (Alanine or other) scanning

• Site-saturation scanning

• Targeted combinatorial mutagenesis

QuikChange HT - Workflow

QC HT Step 1: Design mutagenic library

specific to sequence and application with

eArray design software.

X B C X B C X B C X B C




X B C

X B C

X B C

X B C

X B C

X B C

X B C

X B C

X B C

X B C

X B C

X B C

E.coli library

Screening – Sequencing Identify and sequence

selected clones

Nat

gene V

1 V

2

V

3 V

4

V

5 V

6

V

7

V

N

V1,0

01

Amplified OLS

incorporation

Dpn I digestion

Nick repair

(E. coli)

QC HT Step 3: Perform QuikChange

mutagenesis with each oligo set, move onto

mutant screening in less than a day

Site Directed Mutagenesis Incorporate oligo sets separately into plasmid

DNA with QuikChange, followed by Dpn I

enrichment & transformation

PCR & Purify oligo sets 1,2,3...20

SurePrint Inkjet

Oligo Library

Synthesis

QC HT Step 2: Library is synthesized per design. A set of mutant oligos is

designed for each target region and contains identical 5’ and 3’ ends which serve as

PCR primer binding sites. Oligo sets are isolated from the oligo library by PCR.

Oligo library

cleaved

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FREE design tool: • Supports codon-based design of mutant protein libraries

• Design one pair of PCR primers and one set of mutagenic oligos (“oligo set”) for

each target region; QuikChange HT kits configured to construct up to 10 or 20

sub-libraries (10 or 20 site kits)

• For each oligo set, selections include: • Mutagenesis strategy

• Oligo length / Target size (17 codons with 100mers; 50 codons with 200mers)

• Codon usage

Design Workflow

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eArray Mutagenesis Workspace

QuikChange HT Mutagenesis Strategies

Mutagenic oligos Number unique

per oligo set*

1 mutation type per

codon;

1 codon change per

oligo

17-50

19 mutation types

per codon;

1 codon change per

oligo

323-950

Any of the specified

mutations; 1-4

codon changes per

oligo

Variable

*17-50 codon targets per oligo depending on length (100-200nt);

#Up to 10 or 20 oligo sets per library; 20 oligo sets X 50 codon targets/set = 1000

possible codon targets (~100kDa protein) per QC HT library

Total per library: 120,000#

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Mutation (19 separate codon changes)

50AA x 19 mut = 950 oligos 1 QC HT reaction

Oligo

set 2

QuikScan-19: Site-

saturation scanning

systematically replaces each

amino acid in the wild type

mutational region with all 19

other amino acids, resulting

in 19 mutagenic oligos for

each amino acid position.

For deep mutational

scanning.

Target

sequence

Mutation region 1

Mutation (same codon)

Oligo

set 1

QuikScan-1: Separately

replace each amino acid in

the wild-type mutational

region with a selected amino

acid. Ala replacements

(“alanine scanning”) are

commonly used to identify

key functional amino acids. PCR primer annealing sites

Target

sequence

Mutation region 3

mutations

Oligo

set 3

PCR primer binding sites

QuikCombine: Combine

multiple mutations in

groups of 1-4 positions with

define variation at each

site. Create up to 120,000

variants per library, or

combine a few identified

mutations and validate

functional relevance.

Each oligo library can contain up to 120,000 unique sequences

• create 120,000 different combinations of mutations within a single

domain, or…

• target up to 20 regions in the same protein or different proteins

With 20 sets of oligos, 200nt in length, you can target up to

1000 codons per QC HT library.

11/19/2014

17

1

2

3

4

5

QuikChange HT Workflow

1) Order QuikChange HT kit; includes one custom oligo

library and one pair of PCR primers for each oligo set

2) Amplify the oligo sets separately, and purify using the supplied spin cups:

no need to convert amplicon to ssDNA or remove primer binding sites

3-5) Set up one mutagenesis reaction per amplified oligo

set. Follow the standard QuikChange protocol, substituting

PCR amplicon for the pair of mutant oligos

After transformation and overnight growth, mutant libraries

are ready for sub-cloning into a suitable expression strain

(as needed) for screening or downstream analysis

Sub-library 1 Sub-library 2

Test Cases

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Single Mutation Scanning

11/19/2014

• Before initiating comprehensive site-saturation scanning, we assessed the

mutability of the “fingers domain” of Pfu DNA polymerase

WT FIPSLLGHLLEERQKIKTKMKETQDPIEKILLDYRQKAIKLLANSFYGYYGYA

450 LMTSLLGHLLEERQKIKTKMKETQDPIEKILLDYRQKAIKLLANSFYGYYGYA

451 FIMSLLGHLLEERQKIKTKMKETQDPIEKILLDYRQKAIKLLANSFYGYYGYA

452 FIPMILGHLLEERQKIKTKMKETQDPIEKILLDYRQKAIKLLANSFYGYYGYA

453 FIPRMMGHLLEERQKIKTKMKETQDPIEKILLDYRQKAIKLLANSFYGYYGYA

454 FIPSLMRHLLEERQKIKTKMKETQDPIEKILLDYRQKAIKLLANSFYGYYGYA

455 FIPSLLMNLLEERQKIKTKMKETQDPIEKILLDYRQKAIKLLANSFYGYYGYA

456 FIPSLLGMLLEERQKIKTKMKETQDPIEKILLDYRQKAIKLLANSFYGYYGYA

457 FIPSLLGQMIEERQKIKTKMKETQDPIEKILLDYRQKAIKLLANSFYGYYGYA

458 FIPSLLGHLMEERQKIKTKMKETQDPIEKILLDYRQKAIKLLANSFYGYYGYA

459 FIPSLLGHLLMKRQKIKTKMKETQDPIEKILLDYRQKAIKLLANSFYGYYGYA

460 FIPSLLGHLLEMRQKIKTKMKETQDPIEKILLDYRQKAIKLLANSFYGYYGYA

461 FIPSLLGHLLEEMKKIKTKMKETQDPIEKILLDYRQKAIKLLANSFYGYYGYA

462 FIPSLLGHLLEERMKIKTKMKETQDPIEKILLDYRQKAIKLLANSFYGYYGYA

463 FIPSLLGHLLEERQMIKTKMKETQDPIEKILLDYRQKAIKLLANSFYGYYGYA

464 FIPSLLGHLLEERQKMKTKMKETQDPIEKILLDYRQKAIKLLANSFYGYYGYA

465 FIPSLLGHLLEERQKIMTKMKETQDPIEKILLDYRQKAIKLLANSFYGYYGYA

466 FIPSLLGHLLEERQKIKMKMKETQDPIEKILLDYRQKAIKLLANSFYGYYGYA

467 FIPSLLGHLLEERQKIKTMMKETQDPIEKILLDYRQKAIKLLANSFYGYYGYA

468 FIPSLLGHLLEERQKIKTKMKETQDPIEKILLDYRQKAIKLLANSFYGYYGYA

469 FIPSLLGHLLEERQKIKTKIMKTQDPIEKILLDYRQKAIKLLANSFYGYYGYA

470 FIPSLLGHLLEERQKIKTKMKMTQDPIEKILLDYRQKAIKLLANSFYGYYGYA

471 FIPSLLGHLLEERQKIKTKMKEMKDPIEKILLDYRQKAIKLLANSFYGYYGYA

472 FIPSLLGHLLEERQKIKTKMKETMNPIEKILLDYRQKAIKLLANSFYGYYGYA

473 FIPSLLGHLLEERQKIKTKMKETQMTIEKILLDYRQKAIKLLANSFYGYYGYA

474 FIPSLLGHLLEERQKIKTKMKETQEMIEKILLDYRQKAIKLLANSFYGYYGYA

475 FIPSLLGHLLEERQKIKTKMKETQDPMKKILLDYRQKAIKLLANSFYGYYGYA

476 FIPSLLGHLLEERQKIKTKMKETQDPIMKILLDYRQKAIKLLANSFYGYYGYA

477 FIPSLLGHLLEERQKIKTKMKETQDPIEMILLDYRQKAIKLLANSFYGYYGYA

478 FIPSLLGHLLEERQKIKTKMKETQDPIEKMILDYRQKAIKLLANSFYGYYGYA

479 FIPSLLGHLLEERQKIKTKMKETQDPIEKIMIDYRQKAIKLLANSFYGYYGYA

480 FIPSLLGHLLEERQKIKTKMKETQDPIEKILMNYRQKAIKLLANSFYGYYGYA

481 FIPSLLGHLLEERQKIKTKMKETQDPIEKILLMNRQKAIKLLANSFYGYYGYA

482 FIPSLLGHLLEERQKIKTKMKETQDPIEKILLEMRQKAIKLLANSFYGYYGYA

483 FIPSLLGHLLEERQKIKTKMKETQDPIEKILLD(-)MKKAIKLLANSFYGYYGYA

484 FIPSLLGHLLEERQKIKTKMKETQDPIEKILLDYRMKAIKLLANSFYGYYGYA

485 FIPSLLGHLLEERQKIKTKMKETQDPIEKILLDYRQMTIKLLANSFYGYYGYA

486 FIPSLLGHLLEERQKIKTKMKETQDPIEKILLDYRQKMIKLLANSFYGYYGYA

487 FIPSLLGHLLEERQKIKTKMKETQDPIEKILLDYRQKAMKLLANSFYGYYGYA

488 FIPSLLGHLLEERQKIKTKMKETQDPIEKILLDYRQKAIMILANSFYGYYGYA

489 FIPSLLGHLLEERQKIKTKMKETQDPIEKILLDYRQKAIKMIANSFYGYYGYA

490 FIPSLLGHLLEERQKIKTKMKETQDPIEKILLDYRQKAIKLMTNSFYGYYGYA

491 FIPSLLGHLLEERQKIKTKMKETQDPIEKILLDYRQKAIKLLMNSFYGYYGYA

492 FIPSLLGHLLEERQKIKTKMKETQDPIEKILLDYRQKAIKLLAMTFYGYYGYA

493 FIPSLLGHLLEERQKIKTKMKETQDPIEKILLDYRQKAIKLLAKMIYGYYGYA

494 FIPSLLGHLLEERQKIKTKMKETQDPIEKILLDYRQKAIKLLANSMNGYYGYA

495 FIPSLLGHLLEERQKIKTKMKETQDPIEKILLDYRQKAIKLLANSLMRYYGYA

496 FIPSLLGHLLEERQKIKTKMKETQDPIEKILLDYRQKAIKLLANSF(-)MNYGYA

497 FIPSLLGHLLEERQKIKTKMKETQDPIEKILLDYRQKAIKLLANSFYGMNGYA

498 FIPSLLGHLLEERQKIKTKMKETQDPIEKILLDYRQKAIKLLANSFYG(-)MSYA

499 FIPSLLGHLLEERQKIKTKMKETQDPIEKILLDYRQKAIKLLANSFYGY(-)MNA

500 FIPSLLGHLLEERQKIKTKMKETQDPIEKILLDYRQKAIKLLANSFYGYYGMT

QuikScan-1 Library

Pfu fingers domain consists of 2 anti-parallel helices (O-P)

that make numerous contacts with an incoming nucleotide.

Mutations in this domain have been shown to increase

incorporation of modified nucleotides.

1

2

3

4

5

450 500 IPSLLGHLLEERQKIKTKMKETQDPIEKILLDYRQKAIKLLANSFYGYYGY

# active hits*

*library properties: <10% parental DNA background; total size: 1.7x105 cfus

#95% confidence level; takes into account number of unique sequences and oligo synthesis error rate

• Results show the fingers domain is highly mutable; Pfu DNA polymerase

retains some level of detectable activity when mutated at 34 positions in the

fingers domain

• Heat-treated bacterial cultures were prepared from 467# random clones and

screened for DNA polymerase activity

• QuikScan-1 library containing 51 unique sequences was designed to

introduce a Met (ATG) substitution at each codon in the fingers domain

chromophore

-b

arr

el s

tru

ctu

re

E120G

Site-Saturation

Scanning of GFP

Renilla reniformis GFP E120G:

• Previously identified Gly substitution

at position 120 that improves

fluorescence intensity of Rre GFP in

both E. coli and mammalian cells

• Isolated by random mutagenesis of

humanized Rre gfp sequence and

screening the resulting libraries for

increased brightness in E. coli

• Nucleotide substitution (GAG→GGG)

was subsequently incorporated into

Agilent’s Vitality II (hrGFP II)

mammalian expression vectors

• Resides in 5-6 loop, pointing away

from the chromophore

• Enhances solubility and protein

folding Humanized Renilla reniformis GFP mutant expressed in a range of

mammalian cell types by transfection with Vitality II phrGFP II-C vector

11/19/2014

11/19/2014

GFP Site-Saturation Scan Library Design (expression & screening in E. coli)

Oligo set 1

Strategy: QuikScan-19

Target: 82-231nt / 28-77aa

Oligo length: 200nt

Codon usage: E. coli, R. reniformis

Oligo set 2


Target: 310-459nt / 104-153aa

Oligo length: 189nt


Oligo set 3


Target: 541-690nt / 181-230aa

Oligo length: 198nt


Cloning: Incorporated amplified oligo sets into

pMal-GFP-1 using QC HT kit; pMal-GFP-1

vectors contain frameshift mutations in each

target region; oligos were designed to correct

frameshift (1bp insertion) while introducing

desired codon replacement

Screening: After IPTG induction, fluorescent

colonies were visualized using a light source

and appropriate excitation and emission filters.

Clones displaying higher fluorescence intensity

compared to GFP or GFP E120G transformants

were isolated, and plasmid DNA was prepared.

Positives were subject to 1-2 additional rounds

of transformation and screening to verify

isolation of the brightest GFP mutants.

50AA x 19 mut = 950 oligos 50AA x 19 mut = 950 oligos 50AA x 19 mut = 950 oligos

82 231nt 310 459nt 451 690nt

Screening Results: Domain 1

11/19/2014

Transformation & Screening

(E. coli) AA:

OS1

28-77*

OS2

104-153

OS3

181-230

Library size (x105) 1.3 2.2 1.4

% fluorescent clones 0.5 5.9 7.7

# colonies screened (x104) 1.3 2.5 1.5

No. clones brighter than wild-type 1 14 10

No. clones brighter than E120G 0 8 7

*70-72aa chromophore

• Isolated only one mutation in domain 1 that confers

increased fluorescence

• Domain 1 appears more sensitive to mutations

(0.5% fluorescent) than domains 2 and 3,

presumably due to proximity of the chromophore

N28G

G *

A

V 2

L

I

M 2

F

P

S 1

T

C 1

N

Q 1

Y

W

D 1 1

E 1 2 1

H 1 1 2

K 1

R 1

1

6

11/19/2014

116 120 121 123 124 125 129 131 154

Screening Results: Causal- Brighter than E120G

Comparable to E120G

Putative- (multi-site mutants) Mutations:

Wild-type codon: Key Domain 2

Am

ino

ac

id s

ide

ch

ain

Domain 2

sequence:

5 6 7

Changes to 6 can

modulate fluorescence

G

A

V

L

I

M

F

P 1

S 2

T 1

C

N

Q

Y 1

W

D

E 1

H

K 1

R 1 1

1

6

11/19/2014

Screening Results:

G

A

V

L

I

M

F

P

S

T

C

N

Q

Y

W

D 2

E

H

K

R 1

1

6

G

A

V

L

I

M

F

P

S

T

C

N 1

Q

Y

W

D

E

H

K

R 1

1

6

181 184 185 194 210 213 215 225 229 230

9 10 11

Causal- Brighter than E120G

Comparable to E120G

Putative- (multi-site mutants) Mutations:

Wild-type codon: Key Domain 3

Am

ino

ac

id s

ide

ch

ain

Domain 3

sequence:

“hotspot” in 9

Comparison of Mutagenesis Techniques

Kit Strategy GFP Target

QuikChange HT QuikScan-19 28-77; 104-153;

181-230 (60%)

5x104

GeneMorph II PCR under low-mid-high mutation

rates; EZClone & pool libraries

1-239 (100%) 5x105

Confirmed, isolated as a single mutation

Observations: Most but not all mutants isolated from QuikScan-19

libraries were single-site mutants; allowing the

identification of 17 causal mutations (11 sites) in one

round of screening

• 10 mutations at 7 sites were brighter than E120G

• 7 mutations at 6 sites were comparable to E120G

• 5 putative sites identified in QuikScan-19 clones

containing secondary mutations; non-specified

mutations were attributed to oligo synthesis errors

(1 per >300nt)

Relative fluorescence

of QuikScan-19

mutants:

> wild-type; = GFP E120G

> GFP 120G

Observations cont’d

• In contrast, majority of clones isolated from

EP-PCR libraries were multi-site mutants;

• 4 causal mutations were identified in

single-point mutants; E120G was the

brightest

• 16 putative sites were identified in

multi-site mutants; additional

mutagenesis to distinguish causal from

neutral mutations was not performed

(none were brighter than E120G)

• E120 mutations were common to both

screens (fluorescence emission was

comparable for GFP E120D and E120G)

Key:

> wild-type

= GFP E120G

> GFP 120G

Relative

fluorescence:

QS-19

EP-PCR

Mutagenesis

method:

• 2 of 4 mutations identified by EP-PCR

were located in a region not covered by

site-saturation scanning

11/19/2014

Potential Next Steps

Complete structure-function map of Rre GFP???

Improve fluorescence of mammalian hrGFP expression vectors???

OR

What’s the goal?

11/19/2014

Potential Next Steps Goal: Complete structure-function map of Rre GFP

• QuikScan-19 on remaining domains; site-saturation scanning of the entire

GFP protein could be accomplished with 5 oligo sets (~4500 sequences)

• Assay and sequence a statistically significant number of random clones to

identify not only the brightest mutants, but mutations that eliminate, reduce

or have no impact on fluorescence

2 oligo

sets

Goal: Improve fluorescence of mammalian hrGFP expression vectors

• QuikCombine to create all combinations of brightest mutations (E. coli)

• Domain 2: N116E, E120D, M121E/Q, V123E,

Y124V/C, R125HDK, K129H, R131HS, V154M

• Domain 3: L184RKSTE, M185Y, F194P, G213N,

G229D

Apply site-saturation scanning & rational

combinatorial mutagenesis to 6 and 9

domains, using codon changes that

optimize mammalian expression

Perform in

parallel with

above using

staggered oligo

sets

• Move to mammalian screening system

Agilent SurePrint

Oligo Library

Synthesis

11/19/2014 29

Long length synthesis is achieved by improved

coupling efficiency & lower depurination rates*

2) Oxidation 3) Deblock

1) Coupling

Repeat n times

Depurination

side reaction

N1

N2

Ni O

O P O

RO O

HO

Injet

Flood

O

O

O

O

P O RO

O

P O RO

O

11/19/2014

30

Optimized synthesis chemistry produces high-

quality long oligos

*Synthesis of High-Quality

Libraries of Long (150mer)

Oligonucleotides by a Novel

Depurination Controlled

Process. LeProust, E. et al

(10) NAR 38: 2522–40

PCR

150mer complex library PCR amplified

oligo set

PCR isolates full-length oligos, generating

sufficient material for QC HT mutagenesis

11/19/2014

Long Oligo Advantage

Mutagenize the longest stretch of sequence possible per QC HT reaction

Up to 50 codons with 200mers Up to 17 codons with 100mers

Fidelity of QC HT#

Oligo length 200nt 163nt 112nt

Number clones (bp) sequenced 196 (28.8kb) 198 (21.2kb) 58 (3.5kb)

% correct 63% 70% 81%

% artifacts + wild-type 4% 3% 0%

Number unspecified mutations 84 62 12

Point mutations 4% 8% 0%

Single nt deletions 54% 53% 58%

Deletions of >1nt 42% 25% 34%

Insertion 0% 13% 8%

Oligo error rate 0.29%

(1 per 343nt)

0.29%

(1 per 342)

0.34%

(1 per 294) #PCR errors expected to be <0.2% based on an error rate of 4x10-7MF/bp/d for PfuUltra II DNA polymerase

QC HT Library:

• 93948 unique oligos

• 5 oligo sets (gfp libraries)

• 3 oligos: 112, 163, 200nt

• 2 features per array

Frequency of unspecified mutations is 1 error per ~300 bases; errors are

predominantly deletions (single, multiple)

Trade-off between target length and mutation frequency; longer the oligo, the

greater the likelihood of an unintended synthesis error due to less than

perfect coupling efficiency; simply adjust the number of clones you screen to

take advantage of the benefits of long oligos

Oligo Quality Verified with QC HT Kit Control

• Adapted from standard QuikChange kit control;

processed in parallel with custom oligo sets

• Agilent verifies the quality of each custom oligo library

• 100nt lacZ sequence printed alongside custom oligos

• 10 features per 244K array

• WT lacZ sequence reverts stop codon in pWhiteScript, restoring -gal activity

• Oligo synthesis fidelity verified by scoring percent blue colonies after plating

control QC HT transformations on X-gal/IPTG agar plates

• Researchers can use the QC HT kit control to:

• Confirm oligo library quality

• Troubleshoot issues with protocol (PCR, mutagenesis, transformation) or

reagents (comp cell storage conditions, agar plates)

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Custom

Oligo

Library

Mean %

blue

Std

Dev

1 94.73 2.05

2 92.96 1.91

3 92.00 2.50

4 90.73 3.80

QC HT lacZ kit control

*corresponds to <1 error per 300nt

Quality-controlled oligo libraries

Passing spec: 10l of control transformation

must yield >100 colonies, with at least 80% blue*

General

Guidelines for

Screening

QuikChange HT

Libraries

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Theoretical Screening Requirements

- Assay sufficient number of clones to ensure that most variants have been sampled

at least once

Variants Probability L1 L2

100 0.95 300 760

100 0.99 460 920

1000 0.95 3000 9880

1000 0.99 4610 11510

10000 0.95 30000 122000

10000 0.99 46000 138000

100000 0.95 300000 1448000

100000 0.99 461000 1611000

If the oligo pool contains V variants, the size of library L required to have probability P of observing a particular variant is given by L1 = -V ln(1-P) The size of library required to have probability P that all variants are represented is L2 = -V ln (-ln(P)/V)

Ala scan (50 variants)

Site saturation scan (950 variants)

- A random sampling of 3-10,000 clones should contain at least the majority of

variants in site-saturation scanning library constructed with 200mers;

- Practical number of clones to screen

- Not limited by transformation efficiency of bacteria (~105-107 transformants per

QuikChange reaction), ensuring fully-representative sub-libraries

- Level of oversampling required is determined by the total library diversity

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120K Multi-site (120,000 variants)

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General Screening Strategies

Sub-cloning:

• Purify plasmid DNA library and transform suitable

expression host (BL21, etc.)

Low-throughput screen (1-103): Purified protein needed (e.g., specific activity

changes, activity is undetectable in extracts, etc.)

• Affinity protein purification; process 96 colonies per plate

• Measure protein concentration and functional activity to

detect changes in specific activity or expression level

• Sequence change-of-function mutants to identify useful

mutations; optionally, sequence mutants with wild-type

activity for deeper structure-function mapping

Plate appropriate

number of clones

Transfer to 96-

(deep) well plate;

culture & induce

protein expression

Affinity tag

protein

purification

Measure

specific

activity

Sequence to

identify

essential/non-

essential codons

Availability and type of a mutant screening assay

largely determines mutagenesis strategy

• Low throughput → Alanine scan

• Medium throughput → Site-Saturation scan

• High throughput → Multiple site-saturation scanning

libraries, complex combinatorial libraries

http://2013.igem.org/File:TUM13_Laccase_plate1.jpeg

http://2013.igem.org/File:TUM13_Laccase_Temperature.png

//upload.wikimedia.org/wikipedia/commons/3/3d/Radioactive_Fluorescent_Seq.jpg

Mid-throughput screening

assays (103-106)

Screen recombinant proteins in host cell

background, as extracts (96 well plates)

or filter-lifts (plaques, lysed cells)

Assay activity:

• Directly:

• radiolabelled or colorimetric

substrate (e.g., polymerases, -gal)

• fluorescence (e.g., GFP)

• Indirectly:

• GFP or -gal fusions

• Monitor stability/solubility changes;

expression-level changes in codon

optimization studies

Plate desired

number clones

Incubate with

radio-labeled

substrate

Replica plate

onto filters

Lysis

Isolate clone

from master

plate and

sequence

Identify

beneficial

mutations

Colony-based filter lift

Qualitative

(GoF)

42C 55C

wt wt Mutant polymerase library

Extract screen Example (MMLV RT):

Arezi et al (09) Nucleic Acids

Res., 37: 473-481

Example (T.sp. jdf-3 pol B): Arezi et al (02) J. Mol. Biol. 322:719-729

Selection methods (>106): Viability of recombinant bacteria

under selective metabolic pressure

Binding under stringent conditions

(M13 phage display; e.g., IgG)

Fitness under selective PCR

condition (CSR)

Compartmentalized

Self-Replication

Examples (PCR enzymes):

Hansen et al (11) Nucleic Acids Res. 39:1801-1810;

Arezi et al (14) Frontiers in Microbiology 5: 408

Adapted from

Ghadessy (01)

PNAS 98:

4552-7

Selection

(GoF)

Thermal activity assay:

Colonies were cultured in

deep-well plates overnight.

Cells extracts were prepared

and spotted onto filter paper.

Filters were incubated at

elevated temperature with

primed DNA and radioactive

nucleotides. Mutations that

confer thermal resistance

were identified by DNA

sequencing.

Semi-quantitative

G/LoF

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//upload.wikimedia.org/wikipedia/commons/3/3d/Radioactive_Fluorescent_Seq.jpg

Summary

11/19/2014 38

Evolution of QuikChange Kits

1992 1994 1996 1998 2000 2002 2004 2006 2008 2010 2012 2014

fusion

Accuracy (x105)

PfuUltra Pfu Phusion DV Vent KOD

Pfx Taq

QuikChange

HT Protein

Engineering

System QuikChange

Lightning Site-Directed

Mutagenesis System

QuikChange

Lightning Multi Site-Directed

Mutagenesis System

QuikChange

II & II XL Site-Directed

Mutagenesis System

QuikChange

Multi Site-Directed

Mutagenesis System

QuikChange Site-Directed

Mutagenesis System

Pyrococcus

furiosus isolated

Cloned Pfu

Native Pfu

First of 6

QuikChange

patents issue

PfuTurbo

PfuUltra PfuUltra II

Standard oligos

(1 pair per mutant)

SurePrint Oligo Libraries

(expanded target

regions; up to 50 codons

per reaction)

Standard or

Degenerate oligos

(2-5 per reaction)

Double-to-pentuple

mutants & codon-

saturation libraries

Scanning & targeted-

combinatorial

libraries (up to

122,000 variants)

Parallel improvements in High-Fidelity

PCR & Long-Oligo Synthesis

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QuikChange HT provides fast, efficient, and cost-effective construction of

rationally-designed libraries, enabling comprehensive mutagenesis with

flexible scaling and no prior knowledge of structure-function relationships

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40

• Fast & efficient: 1-day protocol for 3-step cloning with >80% incorporation efficiency

Summary

• Rational design:

• High-quality information content minimizes amount of screening required

• User-specified codon usage eliminates issues with degenerate codon primers;

libraries lack termination codons and biases associated with codon redundancy,

while utilizing host-preferred codons for maximum expression

• Cost-effective:

• In-house method provides lower cost-per-mutant and faster turn-around-times

compared to commercial gene variant libraries

• Use SurePrint inkjet technology to synthesize high-quality oligos up to 200nt in length

• Enables the construction of complex plasmid DNA libraries with targeted mutations

spanning 17-50 amino acids

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41

• Comprehensive; only commercial kit that enables:

• Scanning for deep structure-function mapping with no prior knowledge

• Targeted combinatorial mutagenesis for protein engineering

Key Features cont’d

- Nucleotide-based oligo library design (promoter structure-function studies)

- Kits for incorporating more than one oligo set per QC HT reaction, to

increase library diversity and mutational target size

Future Offerings (potential collaboration and beta-testing opportunities):

• Flexible scaling:

• Easy-to-use mutagenesis design software supports multiple design options

• Construct up to 20 libraries in parallel, targeting one or more proteins

• Choose design strategy that fits experimental and screening requirements, from

alanine scanning to site-saturation scanning and rational combinatorial mutagenesis

Contact info: [email protected]

mailto:[email protected]

Ordering Information

42

All Inclusive:

Mutagenic Primer Library + Library Amplification and Cleanup + QuikChange

Lightning cloning (fast protocol) + Competent Cells + Controls

1. QuikChange HT Mutagenesis Library (one tube)

2. QuikChange HT Mutagenesis Sub-Library Primers (2-40 tubes)

3. QuikChange HT Mutagenesis Reagents

4. QuikChange HT DNA Cleanup Kit Room Temperature (bind & wash buffers + spin cups)

5. SoloPack Gold Supercompetent Cells (15 tubes + 1pUC18)

Scope PN Description US List Price

Commercial G5900A QuikChange HT Protein Engineering System 150nt, 10 sites 10,161.30

G5900B QuikChange HT Protein Engineering System 150nt, 20 sites 15,632.77

G5901A QuikChange HT Protein Engineering System 200nt 10 sites 12,391.83

G5901B QuikChange HT Protein Engineering System 200nt 20 sites 19,064.35

Academic/ government

G5902A QuikChange HT Protein Engineering System 150nt-Academic, 10 sites 6,021.51

G5902B QuikChange HT Protein Engineering System 150nt-Academic, 20 sites 9,263.86

G5903A QuikChange HT Protein Engineering System 200nt-Academic, 10 sites 7,343.31

G5903B QuikChange HT Protein Engineering System 200nt-Academic, 20 sites 11,297.39

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site saturation scanning for every lab: supercharging site ... saturation... · scanning for every...

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