# Presentation: 526902

## 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

## 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

## Slide 10

## How to design oligos

- reverse-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 Protocol

| Oligos  | 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 Protocol

| PCR 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 |  |

## Slide 35

## 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)

## Errors

*p* = mutation rate / 1000 nt / duplication (Cline *et al*., *Nucleic *
*Acids Res 24* (1996))
*Taq* polymerase = 0.008
*KOD* (Novagen) = 0.0027
*PfuUltra* (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

## Errors

- The 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?)