Team:Bielefeld-CeBiTec/Project/unnatural base pair/challenges

• Aim of the Project
• Unnatural Base Pair ▼
• Translational System ▼
• Toolbox ▼

• Overview
• Unnatural Base Pairs
• Uptake and Biosynthesis
• Retention System
• Challenges

• Overview
• Translation Mechanism
• Library and Selection

• Overview
• Analyzing
• Photoswitching
• Labeling
• Photolysis
• Fusing

• Results Overview
• Unnatural Base Pair ▼
• Translational System ▼
• Toolbox ▼
• Parts ▼
• Achievements

• Overview
• Uptake
• Biosynthesis
• Retention System
• Development of New Methods

• Overview
• Translation Mechanism
• Library and Selection

• Overview
• Analyzing
• Photoswitching
• Labeling
• Photolysis
• Fusing

• Parts
• Basic Parts
• Composite Parts
• Part Collection
• Improve

• Hardware
• Software
• Modeling
• Measurement
• Applied Design
• InterLab Study

• Overview
• Gold
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• Public Engagement

• Members
• Advisors
• Bielefeld
• CeBiTec
• Bielefeld University
• Attributions

• Methods
• Oligonucleotides
• Labjournal
• Labnotes Generator
• Safety

Challenges

Short Summary

Expanding the genetic code with unnatural base pairs leads to a variety of challenges. Given that all standard lab methods are designed for a four-letter DNA, methods such as PCR, cloning and sequencing have to be adapted. Additionally, bioinformatic software tools such as BLAST do not work with a six-letter DNA or an expanded amino acid repertoire. Therefore, to validate our work, the development of new methods was essential for our project.

Biological Challenges

All organisms share the same four-letter DNA, composed of the two pyrimidine bases cytosine and thymine and the two purine bases adenine and guanin. This composition of the DNA is the result of billions of years of evolution. The entire cellular machinery is designed to work with or to be compatible with this four-letter DNA composition. Therefore, the addition of two new bases to the DNA proves very challenging from a biological point of view. Firstly, the initial integration of unnatural bases into the DNA is based on the synthesis of modified DNA, which is by now available from almost all DNA synthesis companies. The big challenges arise when it comes to the retention and preservation of these unnatural bases. Since the cell detects them as “foreign”, ergo as mutations, a system has to be put in place to prevent the correction of these bases. Furthermore, the chemical stability of the unnatural base pairs has to be considered. The acceptance of the unnatural bases also poses big challenges, since the entire transcription and translation machineries have to be adapted to accept the unnatural base pairs. This can be achieved by directed evolution of the ribosomes and the tRNA/aminoacyl-tRNA synthetases.

Technical Challenges

Given that most bioinformatic tools are designed to work with only four-letter DNA and the 20 canonical amino acids, expanding the genetic code leads to certain challenges in using these tools. Tools such as BLAST are not designed to work with an expanded base and amino acid repertoire. Therefore, these tools need to be adapted to be used with unnatural base pairs. Other problems that have to be considered are the limited possibilities for sequencing unnatural base pairs, a limited range of compatible polymerases and a restricted set of cloning methods. Sequencing of the unnatural base pairs can be achieved using Oxford Nanopore Technology´s sequencing technology. Since Oxford Nanopore Sequencing does not require additional sequencing chemistry, this technology is predestined to be used for the sequencing of unnatural base pairs. An even simpler solution to this problem is our Mutational Analysis Xplorer (M.A.X), an enzyme based system to analyze if an unnatural base pair is present in a specified sequence or if it is mutated to a natural base. The limited set of cloning methods results mainly from the low fidelity of most polymerases regarding the incorporation of unnatural base pairs. Therefore, if possible, PCR free cloning methods should be used.