Team:William and Mary/Human Practices

3G Assembly

Motivation

This year, our team had the opportunity to share our project with several diverse groups of people: a group of middle school medical explorers, our high school summer interns, and the general public who attended our Building with Biology Public Forum. When connecting with each of these groups we shared the basics behind our project and asked for feedback on our idea as well as general opinions about the work.

Medical Explorers

These students were incredibly excited about the idea of eavesdropping in on cellular conversations. During our discussion some students were interested in the precision of our measurements, equating our “listening in” to the dynamics of signals to a game of telephone where details are lost over time. This gave us the chance to talk about our measurement of florecens and our use of both a flow cytometer and a plate reader. Although a little challenging for the younger age group, several of the students were very excited about the way the machines worked.

The other topic that dominated our conversation were the potential applications. The students wanted to know if we could put our edited bacteria into their bodies and still use the same measurements.

Figure 1: Overview of 3G workflow

Golden Gate Stage

In Golden-Gate Assembly, type IIS restriction enzymes are used to cut DNA. Type IIS restriction enzymes are useful in that they cut outside their recognition sites, creating fragments of DNA with no unwanted base pair scars. We use the restriction enzyme BsaI, which recognizes specific DNA sequences (BsaI sites) and cuts outside of these sites, leaving sticky ends that can be ligated together with T4 DNA ligase.

In this stage of assembly, 3G takes advantage of the Cidar MoClo system, in which specific part types are distinguished by their sticky ends. After being cut, each type of part reveals a distinct sticky end on either side. The standard parts used in most synthetic circuits are promoters, 5’ untranslated regions, coding sequences, and terminators. Their MoClo sticky ends are shown in the image below:

Figure 2: Schematic of MoClo Sticky Ends of promoters, 5' UTRs, CDSs and terminators

The unique sticky ends allow for the parts to line up in the correct sequence before being ligated together. In this way, a full transcriptional unit can be created. To prepare for the Gibson step of 3G, unique nucleotide sequences (UNS) are attached to both ends of the transcriptional unit. The UNS on the 5’ end of the transcriptional unit must have a sticky end A so that it can anneal to the promoter’s sticky end. The UNS on the 3’ end has a sticky end E so that it can anneal to the terminator’s sticky end. These sequences serve as a landing pad for primers in the next stage of PCR. They will also be used when combining the transcriptional units on to a backbone in the final stage of 3G assembly.

There are multiple 5’ UNSs and multiple 3’ UNSs, denoted by numbers (ex: UNS 1, UNS 3, UNS 10). This allows us to combine multiple fragments in the Gibson step.

Figure 3: UNSs attached to transcriptional unit at sticky end A and sticky end E