A fluorescence selection method for accurate large-gene synthesis.

Area of Science:

• Molecular Biology
• Synthetic Biology
• Biotechnology

Background:

• Gene synthesis is crucial for biotechnology but is hindered by errors during the process.
• Deletions are the most common errors in synthetic DNA.
• Accurate and cost-effective gene synthesis methods are in high demand.

Purpose of the Study:

• To develop a practical and efficient method for error detection in low-cost gene synthesis.
• To improve the accuracy of synthetic DNA by identifying and selecting error-free molecules.
• To reduce the cost and increase the reliability of synthetic gene production.

Main Methods:

• Constructed vectors containing multiple cloning sites and a Green Fluorescent Protein (GFP) gene.
• Designed the GFP gene to be initially out-of-frame.
• Utilized a fluorescence-based readout to identify colonies with in-frame synthetic DNA inserts, indicating error-free molecules.

Main Results:

• Successfully synthesized five genes using the developed method.
• Achieved a significant improvement in accuracy, increasing the base pairs per error from 629 to 6552.
• Demonstrated the effectiveness of selecting green fluorescent colonies for isolating error-free synthetic DNA.

Conclusions:

• The fluorescence-based method provides a practical solution for detecting errors in synthetic DNA.
• This approach enhances the accuracy and efficiency of low-cost gene synthesis.
• The method has the potential to significantly impact fields relying on synthetic DNA, such as synthetic biology and genetic engineering.