Functional DNA is at the heart of modern biotechnology. At the Great Lakes Biotech Academy our key educational goal is to teach students how to find and create functional DNA. Once a functional DNA sequence is discovered it may be challenging to produce in the lab -- especially if you don't have access to sophisticated (and expensive!) scientific equipment. To address this challenge most of my time in the academy lab the past few months has been focused on finding ways to simplify and reduce the cost of DNA assembly. This post will provide an overview of my progress thus far.
A brief history of DNA assembly
The modern era of molecular biology was enabled by two key inventions: the polymerase chain reaction (PCR) and restriction enzymes. PCR was developed in the early 1980's and allows the facile copying of small to medium sized DNA molecules(typically 100 to 5000 base pairs). With PCR it became relatively easy to amplify and purify many types of functional DNA. Restriction enzymes are part of bacterial immune systems that recognize and cut short sequences of DNA. Some restriction enzymes create "sticky ends" after they cut and these sticky ends can be used to join different pieces of DNA together. For 20+ years most molecular biologists used PCR and a plethora of different restriction enzymes to build functional DNA molecules. The techniques worked, but most DNA molecules required different restriction enzymes and it was especially difficult to build large DNA sequences. In the early 2000's new DNA assembly technologies were developed that allowed much easier synthesis of large DNA sequences. One of these technologies, The Gibson Assembly method, was used to synthesize an entire genome and create a synthetic life form. Gibson Assembly is an extremely efficient way to produce large DNA sequences, but it is inherently expensive because relatively large homology regions must be added to every DNA fragment in the assembly.
Hierarchical cloning and the Golden Gate Assembly method
With Gibson Assembly any large DNA sequence can be synthesized, however the expensive DNA fragments used to assemble the large DNA sequence generally can't be reused for related designs. When assembling multi gene pathways researchers often want to test the effect of different promoters, coding sequences, and terminators for every gene in the pathway and Gibson Assembly is not a cost effective way to explore that parameter space. In 2008 a new method for hierarchical DNA assembly, termed Golden Gate cloning was published by Engler et al. For Golden Gate cloning, the Type IIS subclass of restriction enzymes was used in a clever way to join multiple DNA fragments very efficiently and in the process remove the restriction enzyme recognition site from the product. This approach worked because type IIS restriction enzymes cut outside their recognition sequence and can thus be used to create custom "sticky ends". By nesting two different Type IIS restriction enzymes a molecular biologist can put together larger and larger pieces of DNA. The only caveat of the Golden Gate Assembly method is that every DNA fragment in the assembly can not contain the Type IIS restriction site used in the Golden Gate reaction. The commonly used Type IIS restriction enzymes BsaI, BmsBI, and SapI have either 6 or 7 base pair recognition sites so for most genes this is not an issue.
Recent improvements on the Golden Gate Assembly method have involved exploiting the sensitivity of the BsaI restriction enzyme to DNA methylation and I am incorporating this approach into the biotech academy's molecular biology platform. This will allow us to follow a very simple and low cost procedure to assemble functional DNA several thousand basepairs in size.
progress towards a molecular biology platform
The first experiments related to the molecular biology platform were performed in May and were done to determine if a subset of BsaI sites could be blocked effectively by a DNA methyltransferase. Two commercially available methyltransferases, MspI and HpaII both recognize the sequence CCGG and methylate the external or internal cytosine, respectively. By adding two cytosine bases immediately 5' to the BsaI restriction site a MspI and HpaII methylation site can be created that overlaps the BsaI recognition sequence. The schematic below summarizes the general strategy where the methylation sensitive mBsaI site is on the outside of the insert (on the vector backbone) and the methylation insensitive site is on the interior and linked to a LacZalpha marker.
It was known in the literature that methylation could severely inhibit BsaI, but it hadn't been reported if the methylation pattern catalyzed by either MspI or HpaII could block BsaI. To test this I PCR amplified a portion of the Neurospora crassa genome that contained the mBsaI site. The PCR amplified product was treated with MspI and HpaII and then subjected to digestion with a high fidelity version of BsaI. The methylation by MspI and HpaII appeared to completely protect the DNA from cleavage (see below).
Building the pglb vectors
So the next step was to incorporate this methylation sensitive BsaI site into a useful cloning vector like pUC19. The series of vectors created at the Great Lakes Biotech Academy are called pGLB vectors, where X designates the number of the vector. The design of the pGLB vectors includes blue/white screening using a modified LacZalpha fragment, a modified version of beta-lactamase which reduces the production of satellite colonies, and the incorporation of the methylation sensitive and insensitive BsaI cloning sites. The other modifications in the pGLB vectors will be highlighted in future posts.
After building this vector tests were performed to judge the effectiveness of the cloning strategy. The pGLB2 vector is produced in E. coli like any other plasmid. After miniprepping the plasmid it is treated with HpaII or MspI methyltransferase and then is ready for use in the Golden Gate cloning reaction. At the Great Lakes Biotech Academy we use a modified protocol for Golden Gate Assembly that uses high fidelity BsaI restriction enzyme, T7 DNA ligase (instead of T4 DNA ligase), pGLB2, and the Golden Gate compatible DNA inserts. For the initial test of pGLB2 we performed three component assemblies with the Lac promoter (part 1), a green fluorescent protein gene (part 2), and methylated pGLB2 (part 3).
Possible outcomes for this reaction when the transformed cells are plated onto LB agar supplemented with ampicillin and X-gal are as follows:
- Green fluorescent colonies -- a successful reaction!
- Blue colonies -- a failed reaction, indicating re-ligation of the vector
- White colonies -- a failed reaction, indicating loss of LacZalpha and mis-ligation of vector
- No colonies -- a failed reaction, inserts with improper overlaps
The initial test showed thousands of green fluorescent colonies and just a handful of blue colonies (can you find them in the image?). Control experiments where no GFP insert was included showed no colonies. Together these results suggest that the pGLB2 cloning strategy works very efficiently. Soon we will use this system to rapidly assemble several pieces of functional DNA into a biosynthetic pathway.