FAQ
Where can I find the IUPAC nucleotide ambiguity codes?
Please visit: http://www.chick.manchester.ac.uk/SiteSeer/IUPAC_codes.html
What is the rational behind directed evolution?
In vitro molecular evolution strategies are perhaps the most efficient methods of creating proteins or DNA elements with improved or novel properties. It offers the possibility to screen a huge amount of different variants of a certain gene to select a version that performs better towards a specified goal than the original gene. Essentially it is the transfer of natures principle of the "blind watchmaker" to develop complexity and perfection.
What should a library look like if I want to improve my protein?
This largely depends on the project. In general, it is advantageous to use as much information about the protein as possible: crystal structure, conserved motifs, presence of homologs, to mention just a few. If you are uncertain please contact us and profit from our experience.
Can you give an example of how a designed library looks like?
This is a section of a typical degenerated sequence (red) and a few sequenced clones aligned below (black). Three codons are degenerated by the motif "NNB", which allows for the coding of all 20 amino acids.
What are the advantages of synthetic libraries over conventional protocols for creation of diversity?
Conventional protocols for library creation incorporate a large fraction of unwanted randomness (e.g. error-prone PCR) and strict limitations in disjointing of adjacent SNPs (e.g. DNA shuffling). Synthetic degenerated libraries limit the introduction of mutations only to those sites and with the exact frequencies that are determined by the scientist. In addition, adjacent SNPs will be shuffled independently of their proximity. It is the method of choice to create rational diversity.
Why do I need rational diversity when I may screen a large number of random mutants anyway?
Because the number of possible variants of a protein are astronomic and exceed the capacity of even high-throughput screenings by many orders of magnitude. On the other hand, any laboratory technique allows only for the screening of a limited library size. A library created by introducing randomness by chance will only represent a tiny random fraction of all possible variants. This is one reason why it is smart to carefully design the library considering reasonable predictions and to synthesize a library which contains exactly and only these variants. Here is a more detailed description about this topic.
What types of libraries can be produced by GENEART?
Combinatorial Libraries (up to 1011 variants)
degeneration of multiple codons
Site-Saturation Mutagenesis (up to 20 variants)
degeneration of one single codon
Sequential Permutation Libraries (# of codons x 20 variants)
successive site-saturation mutagenesis
Controlled Randomization (up to 1011 variants)
unbiased random substitutions with defined frequency
SH3 Domain Phage Display Library
cloned cDNA of all known human SH3 domains
Truncation Libraries
customer defined truncations without out of frame mutations.
What material must be supplied for a library synthesis?
No material at all. All we need for library creation is the sequence file, submitted electronically, and information about the position and nature of the degenerated sites. You may use our online quote to get a first estimate of the cost of your project. Detailed information about the library is then usually specified via direct communication between the scientist and GENEART scientists prior to the project.
Can a library be subcloned in my vector?
Yes. For this purpose customers have either sent their own, individual vector or advised us to clone the library into a commercially available vector.
How will a library be delivered?
Many different options are available:
A library can be delivered as a linear DNA fragment, ready for digestion and subcloning. It can be subcloned by GENEART into any vector and will then be delivered as plasmid preparation, amplified one round through transformation and plating of E. coli. Additionally, the transformed cells can be sent as a glycerol stock. Individual, sequence verified clones can be sent on plate or as glycerol stock. In the case of subcloning into phage display or yeast two-hybrid vectors, phage supernatants or transformed yeast strains can also be delivered. All libraries come with an extensive documentation and a statistical analysis of a sequenced peer group of single clones.
Please provide some examples where directed evolution might help?
- Increase or adjust promoter strength or specificity
- Enhance or modulate protein stability
- Modify or combine enzyme properties
- Increase binding affinities of receptors, ligands and antibodies
- Optimize or alter signal peptide efficiencies
- Destroy protein function while retaining immunogenicity
- Combine and select natural polymorphisms
- Increase protein half life
- Adjust thermal stability
What quality criteria can GENEART guarantee?
Three criteria are important for degenerated libraries and can be guaranteed at various levels:
- Maximum correctness of the non-degenerated parts
- Maximum variation of the degenerated positions with even nucleotide distribution
- Maximum size of the total library
Does GENEART offer screening services for the produced libraries as well?
We are aware that many labs are not equipped to carry out high throughput library screenings. We offer several systems to identify the best candidates of a library. For specific information, please inquire.
What are the major benefits for the customer?
- No need for any template. Synthesis from ASCII file.
- All control over every important feature of the library. Create rational diversity.
- Significantly reduced screening efforts over conventional methods.
- Generate combinations, not possible by conventional methods.
- Guaranteed maximum correctness, maximum variation and maximum size.
- Greater chance of successfull screening.
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