Overview
This year, we took the lead in using a new DNA assembly strategy in iGEM – the YeastFab technology,
which can ensure efficient multi-gene assembly. Using this technology, we have built a large number of
parts, including S. cerevisiae promoters with different strengths, different terminators, ORFs,
homologous arms and selection markers, which can help researchers to complete efficient constructions.
Our optimized YeastFab can make the construction process more standardized and modular. Unified systems
and procedures, and high positive rates can greatly reduce the use of instruments, and simplify the
operation procedure. We verified the feasibility of this DNA assembly strategy by integrating an exogenous
lycopene synthesis pathway into the genome of S. cerevisiae with the final product lycopene detected by
HPLC. We also developed a corresponding hardware operating platform to manifest this technology. Relying
on mechanical automation, well standardized and highly efficient DNA assembly process, we can quickly
assemble a large number of DNA fragments in a short time. We hope to build an automated standard DNA
assembly platform, and make it open to all iGEM teams around the world, with the aim of making more
iGEM teams enjoy a simplified construction process, and improve their construction efficiencies.
Construction method
Currently, conventional construction methods have many disadvantages, such as time-consuming digestion and ligation, and extraction of linearized backbone plasmid vectors with unsecured purity and quantity. Sometimes, we even have to make a lot DNA preparations, and are always troubled by self-ligation of digested vectors. What is more the ligation mostly requires overnight incubation, and the efficiency and quality may not always meet our satisfaction. Using the YeastFab technology, we can take the advantage of two IIS restrictive endonucleases Bsal and BsmBI through the high-fidelity and efficient Golden Gate method, with the recognition sites of the enzymes added to both ends of a target gene. Because the recognition site and cleavage site of an IIS type restricted enzyme is separated, we can specifically design sticky ends based on this method. Thus, a modular part library can be generated within a week with a success rate of nearly 100%. We designed the pccdK2 and TU series vectors based on the YeastFab principle. These two types of vectors have recognition sites of BsmBI and BsaI，but the digesting regions of the two enzymes are placed in opposite positions between the two sets of vectors. After rational design of the cleavage sites among the vector of the same series, the reverse complementary sticky ends can be successively obtained under the action of the corresponding type IIS restriction enzyme (see in Design page for detail). These parts can be easily characterized and assembled into transcription units (TUs). Using this efficient DNA assembly approach, we successfully integrated the synthesis pathway of lycopene into the genome of S. cerevisiae and verified its production through phenotype determination, HPLC analysis, and PCR amplification of extracted genomic DNA.
Figure 1. The constructions of the basic parts (Promoter, ORF and Terminator)
Hardware
Based on our efficient DNA assembly strategy, we have independently built a cost-effective and practical hardware platform, which can realize three basic operations of pipetting, temperature control and colony picking. The modular design also makes the platform more practical. After preparing the samples needed for construction, we can add them to the system through a liquid transfer arm. The temperature control board can not only ensure a relatively cold environment for enzymes, but also achieve the temperature required for the reaction. Compared with ordinary liquid transfer stations, our device may not only be visually recognized by a build-in camera, but also accurately classify colonies of different colors as needed. Meanwhile, we added the function of colony size discrimination and positioning. The algorithm can give the central coordinates of each colony, and select the ones with an appropriate size based on actual needs. Through physical distance conversion, the colonies can be selected by a hardware part, which includes all the operations needed to build the experiment, and increases the degree of automation to a new level. Importantly, the price of this hardware platform is less than $1,000, which provides a cost-effective solution for synthetic biology enthusiasts, and is suitable to be promoted to more iGEM teams and synthetic biology enthusiasts. During the implementation period, due to too many signal lines connected by the circuit board, it was easy to drag to some signal lines during the operation of the frame, resulting in poor contact or even damage of the signal lines. To solve this issue, we wrapped the wires with an envelope tube, so that the original dozens of wires were merged into several clusters. As a result, the whole hardware was more compact, and the security risks were also reduced. Finally, we would like to share our hardware construction plans and operation manuals with other iGEM teams to promote synthetic biology. We hope that this hardware platform can enable more iGEM teams to have efficient construction solutions, so that more people can participate in synthetic biology through this economical and efficient hardware platform.
Figure 2. Diagram of the hardware platform
