Golden Gate

Overview

Golden Gate assembly is a technique of cloning that allows for the simultaneous assembly of multiple DNA inserts into a single vector through the use of Type IIS restriction enzymes (BsaI) (1). This is done by designing overlapping ends (Type IIS restriction sites) which can join the DNA inserts together (1). Although Cellucoat’s proof of concept was done with nisin, nisin has limited effectiveness against fruit fungi, as shown by the results collected through our partnership with U of Alberta iGEM. When implementing our fruit preventive coating into industry, we want stakeholders to be able to customize their BC to meet their particular needs in regards to the pathogens it is resistant against.

The seamless cloning that golden gate offers will allow for this customization through the use of the RFP flipper device, allowing nisin to be swapped for other antifungal peptides. The red fluorescent protein (RFP) golden gate flipper device (BBa_K3114015) consists of an RFP coding device, which is flanked by inverted Bsal recognition sites (our restriction enzyme of interest) as well as fusion sites. This can then be utilized in converting parts designed for Type IIS assembly into standard BioBricks. The advantage of using our RFP flipper system is that the successful assembly reactions that use this part will substitute the RFP coding device with the Golden Gate inserts; successful assembly products can be screened by their white color while unsuccessful assembly products will be red in appearance.

Using this workflow, we worked towards modularizing our antimicrobial peptide expression for Cellucoat’s customizable preservative properties.

Proof of Concept

To demonstrate the applications of Golden Gate assembly in modularizing our expression system, we set out with two objectives:

1. Isolating 2019 Team Calgary signal peptide constructs for Golden Gate assembly, with an end goal of using them in the Golden Gate assembly procedure.
2. Insert recombinant nisin (Nisin Q and Nus A) into the RFP flipper using Golden gate assembly.

Results

For the first proof of concept objective, our team was able to perform a restriction enzyme digest on the 2019 signal peptide sequences and verify this through an electrophoresis experiment (Figure 1). This experiment did not proceed to isolating the signal peptide sequences due to difficulties with PCR amplifying the signal peptide insert.

Figure 1. Lane 1 contains 2uL of Quick-Load Purple 2-log 1kb plus ladder. Each lane contains 10uL of the digested sample of various signal peptides. Prepared on a 1.5% gel which ran at 100V for 50 minutes.

In regard to the second object of inserting our recombinant nisin insert into the RFP flipper, we were able to amplify and successfully insert what we predict to be our nisin insert from G-Block 2 (NisQ with an N-terminus NusA solubility factor and 6x His-tag) in Figure 2.

Figure 2. The visualization of the insertion of recombinant nisin into the RFP flipper via golden gate assembly. Lane 1 contains Quick-Load Purple 2-log 1kb plus ladder, lane 2 contains G-Block 4, and lane 3 contains G-Block 2. Products were visualized in 1.2% agarose gel at 100V for 50 minutes.

Future Directions

Our proof of concept involved inserting nisin into the RFP flipper system, demonstrating the effectiveness of our restriction enzyme. In the future, we aim to swap out nisin for one or more antifungal peptides and include various signal peptide constructs. Potential antimicrobial peptides include the cyclic lipopeptide Iturin A, the proline-rich peptide Bac5, Pumilacidin (2). A major barrier in making this modular expression system come to life is promoting the testing, development, and approval for antimicrobial peptides other than nisin, as it is currently the only food safe antimicrobial peptide.

References

1. Engler C, Marillonnet S. Golden Gate cloning. Methods Mol Biol. 2014;1116:119-131
2. Fernández de Ullivarri M, Arbulu S, Garcia-Gutierrez E, Cotter PD. Antifungal peptides as therapeutic agents. Frontiers in Cellular and Infection Microbiology. 2020;10.