Gene Synthesis using DNAWorks

Dr. David Hoover

Helix Systems, SCB, CIT, NIH

Gene Synthesis

Several methods• ligation - incredibly tedious and inefficient• FokI - sequence dependent (type IIs r.e.)• serial cloning - sequence dependent• assembly or self-priming PCR

Gene Synthesis Methods

Thermodynamically Balanced Conventional

Thermodynamically Balanced Inside-Out

Protein Expression

Protein/Structure Independent Factors:• promoters and upstream elements• translational initiation and termination• mRNA stability• codon bias

Protein/Structure Dependent Factors:• folding and aggregation• proteolysis and degradation• secretion and localization

Codon Bias

0.0%

10.0%

20.0%

30.0%

40.0%

50.0%

60.0%

70.0%

80.0%

90.0%

R:A

GG

R:A

GA

I:A

TA

G:G

GA

P:C

CC

R:C

GA

L:C

TA

R:C

GG

T:A

CA

L:T

TA

S:A

GT

S:T

CA

L:C

TG

S:T

CG

R:C

GC

R:C

GT

A:G

CG

P:C

CG

E. coli

H. sapiens

Synthetic Genes

Benefits:• Codon use optimized for host• Flexibility in subcloning• Ease of complex mutagenesis

Problems:• Time consuming• Complicated• Error-prone

Commercial Sources

Blue Heron Biotechnology (http://www.blueheronbio.com)

DNA 2.0 (http://www.dna20.com/)

Gene Script Corporation (http://www.genscript.com/)

BioNexus Inc. (http://www.genesynthesis.net/)

Entelechon (http://www.entelechon.com/)

GeneArt (http://www.geneart.com/)

Codon Devices (http://www.codondevices.com/)

Commerical Sources

Typical costs:• $0.79 - $3.60 / bp• Complexities?• Intellectual property?

• 800 bp = $1000 (Gene Script)

Genes From Scratch

• oligos ~ $0.20 / nt (NIH discount)• PCR reagents ~ $2 / reaction • sequencing ~ $20 / 600 bp• electrophoresis ~ $3 / gel• labor ~ $20 / hr

GFP, 238 aa, 714 bp, 20 oligos, 1134 nt, 2 reactions, 2 gels, 4 sequences, ~10 hrs = $517

How to design oligosreverse-translate protein into DNA, optimum codon

usage

break into fragments of equal overlap Tm

optimize:• hairpins / mRNA structure• repeats / mispriming• restriction site inclusion / exclusion• length

DNAWorks

http://helixweb.nih.gov/dnaworks/

DNAWorks Output

181 TCTGGTGAAGGCGAGGGTGACGCGACCTACGGTAAACTCACTCTCAAAT agaccact TGCCATTTGAGTGAGAGTTTAAGTAGACGTGG <--- 4 S G E G E G D A T Y G K L T L K F I C T

| | | | | | |

7 ---> 241 ggttccttggccgaccctggttactaccttctcttacggtgttcag TGCCCGTTTGACGGCCAAGGAACCGGCTGG tc <--- 6 T G K L P V P W P T L V T T F S Y G V Q

| | | | | | |

DNAWorks Options

• Job Name• E-mail Address

DNAWorks Options

Codon Frequency Table• E. coli (standard, class II), H. sapiens, C.

elegans, D. melanogaster, M. musculus, P. pastoris, R. norvegicus, S. cerevesiae, X. laevis

• Custom CFT

Gly GGG 599428.00 16.49 0.25 Gly GGA 597986.00 16.45 0.25 Gly GGT 392298.00 10.79 0.16 Gly GGC 814464.00 22.41 0.34 Glu GAG 1441162.00 39.65 0.58 Glu GAA 1043166.00 28.70 0.42 Asp GAT 789799.00 21.73 0.46 Asp GAC 914677.00 25.16 0.54 Val GTG 1028789.00 28.30 0.46 Val GTA 257442.00 7.08 0.12 Val GTT 399567.00 10.99 0.18 Val GTC 528840.00 14.55 0.24 Ala GCG 271820.00 7.48 0.11 Ala GCA 579156.00 15.93 0.23 Ala GCT 672416.00 18.50 0.26 Ala GCC 1018345.00 28.02 0.40 Arg AGG 432954.00 11.91 0.21 Arg AGA 434655.00 11.96 0.21 Ser AGT 441137.00 12.14 0.15 Ser AGC 706723.00 19.44 0.24 Lys AAG 1163126.00 32.00 0.57 Lys AAA 879684.00 24.20 0.43

DNAWorks Options

Parameters• Annealing Temperature• Oligo Length (random)• Codon Frequency Threshold (random, strict,

scored)• Oligonucleotide, Na+/K+, Mg2+ Concentrations• Number of Solutions• TBIO• No gaps in assembly

DNAWorks Options

Balancing act• Fast, simple, cheap?• Slow, complex, expensive? - reliable• Reusable and interchangeable oligos?

DNAWorks Options

Others• Restriction Site Screen (non-degenerate,

degenerate sequences)• Custom Site Screen (mind the format!)• Weights (experimental)

DNAWorks Options

Sequences• protein (X = stop)• nucleotide (can be degenerate)• almost any file format• reverse sequence• fix sequence in gap

DNAWorks Output

Web output• Input for DNAWorks (standalone version)• Header• Initial parameters• Optimization log• Final scores• Final summary

DNAWorks Output

Total output• Sequence blocks• CFT blocks• Pattern block• Trials• Final Summary

DNAWorks Output

Trial outputs• Initial parameters• Final DNA sequence• Assembly• Final scores• Codon report• Histograms• Oligo sequences

Scores / Penalties

• codon usage• length• melting temperature• repeat• pattern• mispriming• AT/GC contents• gapfix

Mutant Run

• Design oligos based on previous set of oligos• Parameters taken from previous run• For single mutation, will output 1 or 2 oligos only

What to look for

Final Summary• Avoid misprimes and repeats• Make sure overlaps are > 12 nt (Short)• Tm range should not be > 3°C (TmRange)

Don't depend entirely on scores• Arbitrary, somewhat dependent on length

Tricks

Choosing codons• random - slower optimization, less constrained• strict - for the fussy• scored - if codon score really matters

Tm, Length ranges, Number of Solutions• To find the "very best" solution• no more than 999

Tricks

Design multi-use and interchangeable oligos• Flanking primers with standard overlaps• Intersperse nucleotide elements between protein

elements• Gap-fix restriction sites• Allow for mutations later on

Random mutagenesis• Nucleotide sequences can be degenerate

Tricks

Thermodynamically Balanced Inside-Out Mode• Multi-step PCR• More controlled, reliable method• Gao X., et al., Nucleic Acids Res 2003

Random oligo lengths• Faster, better optimization• For the not-so-fussy• Probably best for DNA-only genes

Tricks

Set Tm higher• 64°C - 70°C• longer oligos, extra purification ($$$)

Always double check!

Nothing is foolproof• Think carefully about what you need BEFORE

starting work• Always run final sequences through alternate

program (EMBOSS, GCG-Lite)• Make sure oligos are what you intended

PCR

• Mix all oligos and additives• Specific PCR protocols• Analytical gel• Isolate desired products

Assembly ProtocolOligos 1 μl 625 nM each 25 nM each

dNTPs 2 μl 2.5 mM each 0.25 mM each

H2O 19 μl

Buffer 2.5 μl 10X 1X

Pfu pol. 0.5 μl

95°C 2.0 ' 1X

95°C 0.5 '

65>55°(-0.5) 0.5 ' 20X

72°C 0.5 '

72°C 5 ' 1X

4°C hold

Amplification ProtocolPCR mix 2 μl ? ?

dNTPs 8 μl 2.5 mM each 0.2 mM each

3' primer 4 μl 10 μM 400 nM

5' primer 4 μl 10 μM 400 nM

Buffer 10 μl 10X 1X

H2O 70 μl

Pfu pol. 2 μl

95°C 2.0 ' 1X

95°C 0.5 '

62°C 0.5 ' 20X

72°C 0.5 '

72°C 5 ' 1X

4°C hold

Problems

• No product (complete failure)• Wrong size product (mispriming)• Mutations (2 out of 3 correct, 2 errors/kb)

Sequencing is warranted...

Fixes

• Optimize PCR conditions• Break gene synthesis into steps (TBIO)

Errorsp = mutation rate / 1000 nt / duplication (Cline et al., Nucleic Acids Res 24 (1996))

Taq polymerase = 0.008KOD (Novagen) = 0.0027PfuUltra (Stratagene) = 0.00043

The probability of a gene n bp in length having no errors using a polymerase with mutation rate p:

p' = (1 - p)n

Therefore, p' for a 738 bp gene = (1 - 0.00043)738 = 0.728

ErrorsThe number of clones needed to screen to find a correct gene with 95% confidence:

N = log(0.05)/log(1-p')

Thus, log(0.05)/log(1-0.728) = 3 clones need to be sequenced.

From Wu et al., J Biotech 124 (2006)

Time

• Find protein of interest, design oligos, order oligos

• Run PCR, integrate into sequencing vector, transform

• Pick colony, grow overnight culture

• Miniprep construct, integrate into expression vector, transform

• Pick colony, grow overnight culture

• Run expression growth trials

~ 1 week between concept and initial trial (at best!!)

Can be automated and parallelized (96 well plates?)