Codon-precision mutant libraries
Codon-precision gene mutant libraries are based on sets of codons for each randomized amino acid position. In contrast to libraries generated using randomized single nucleotides, codon-precision libraries avoid undesired codon variants or stop codon, thus reducing the cost of screening significantly. The libraries are based on a new method developed at Entelechon.
- No undesired codon variants
- No stop codons
- High precision synthesis prevents frame shifts and mutations
Conventional libraries suffer from the problem that the undesired codon variants lead to redundant protein variants with a low randomization efficiency. Due to the redundant codons, some amino acids are overrepresented simply because they are encoded by more synonymous codons than others.
Entelechon’s codon-precision libraries avoid these problems. One or more amino acid residues are randomly substituted – by a specific codon for all twenty biogenic amino acids or for subsets defined by the customer. The codons can be mixed in any desired ratios (starting at 1% per individual codon). Multiple randomized positions can be used in the same gene sequence. Quality control ensures the correct codon distribution and the absence of undesired mutations or frame shifts.
Example applications:
- Protein engineering
- Alanine scanning
- Antibody development
- Enzyme design
Advantages
Since a codon-precision library avoids undesired codons, the overall complexity of the library is reduced significantly. This saves time and work during screening.
Reduced library Complexity
| Conventional | Entelechon | |
| Codons | 32 | 20 |
| Stop codons | 1 | 0 |
| Minimal # of codons/aa | 1 | 1 |
| Maximal # of codons/aa | 3 | 1 |
| Example: Total number of variants for the library with 6 randomized codons |
326= 1.07 x 109 |
206= 6.4 x 107 |
As you can see, for a library containing six randomized codon positions, Entelechon’s technology reduces the complexity significantly and makes sure that you only screen variants you are really interested in.
In addition, due to the degeneracy of the genetic code and the fact that some amino acids are encoded by more codons than others, the conventional library will overrepresent some amino acids threefold as compared to others. A codon-precision library avoids this problem. This translates into significantly reduced hands-on time when screening a library.
Reduced time for screening the library
Highly efficient screening with FACS equipment allows to screen 107 events per hour. For the above mentioned example of a library with 6 fully randomized amino acid positions this converts into:
| Conventional | Entelechon | |
| Required man-hours | 107 hr (13 work days) | Just 6 hours |
Entelechon reduces a 2-3 weeks project just to one day screening.
Deliverables
The library can be delivered as double stranded free DNA, or inserted into a cloning vector.
Optionally, a culture of the host cells (E. coli) can be delivered.
The maximal number of obtainable clones varies with the gene, vector and host cells, but is usually in the range of 107 to 109.
How to get a codon-precision library
We will help you designing the optimal library for your project. Please visit our contact page for our contact details.
If you are unsure about the design of a mutant library, please ask us. We can advise on the number of randomized residues, on the codon distribution, and other experimental features. Depending on the underlying problem, multiple iterations of constructing a library and screening may be required to achieve optimal results.
Entelechon’s codon-precision libraries come royalty-free, and Entelechon does not claim any rights to products based on these libraries.