Engineering Success
The Engineering Design Cycle
Motivated by the increasing demand for novel solutions to antibiotic resistance, our team set out to build a state-of-the-art biological system that utilizes synthetic biology principles to address the worldwide challenge of antibiotic resistance. Our approach follows the foundational engineering cycle of design, build, test, and learn.
Cycle 1: Learn
Starting this project was an interesting challenge and a great way for our team to learn a lot about synthetic biology, as we are not experienced with it. We were keen to investigate how biology could be constructed in the same way as machines, utilizing DNA as building blocks to address practical challenges. To begin, we thoroughly studied the fundamentals of synthetic biology, including the assembly, regulation, and optimization of genes. As we learned, we applied these concepts to design a system that addresses antibiotic resistance; “Thermoguard” a novel antimicrobial peptide that targets staphylococcus aureus and could be applied for the treatment of skin and soft tissue infections. Along the way, we encountered many hurdles, but with each challenge came new insights that shaped our design into what it is today.
Cycle 2: Design
After a rigorous Screening of fifty thermophilic bacterial isolates for their ability to produce antimicrobial peptides AMP. We finally chose Brevibacillus borstelensis AK1. We had to do several bioinformatics analyses to identify our novel genes of interest before developing our circuit. We discovered six genes in our bacterial genome that match our query sequences using an alignment-based method.
In our initial learning phase, we found that AMP genes often come in clusters within the bacterial genomes. This cluster includes genes responsible for the synthesis, processing, immunity, and export of bacteriocin. We went further to use software like antismash and interpro (https://www.ebi.ac.uk/interpro/search/sequence/) to find these sequences. For more details, please refer to the Bioinformatics page.
Verifying if these genes were AMP genes was an important aspect of our project. Hence, we had to first express our predicted AMP genes in an appropriate host, confirm that the peptides are being produced and that these peptides processed antimicrobial effect on our target strain, Staphylococcus aureus.
We design a basic genetic circuit to optimize the expression of our AMPs. Our plan was to clone our genes, firstly using a single plasmid system containing a predicted AMP gene, then a two-plasmid system: one containing a predicted AMP gene and the second containing the asfR gene.
The asfR gene encodes for a transcriptional regulator that influences the production of secondary metabolites in bacteria. Since this gene was found in the genome of our thermophile, we wanted to know if it had an influence on the production of AMPs.
Our synthetic genes of interest were optimized for expression in E. coli. We employed the type IIS cloning technique to create a composite part by joining four fundamental components to create the vector carrying our AMP genes. Each composite part contained a strong promoter, i.e. AB_pTac promoter which does not require any special RNA polymerase (BBa_J435360); an E. coli codon optimized RBS (BC_BCD12) (Part BBa_J435305), the AMP coding sequence and a terminator containing a TEV protease recognition domain, GFP, and His tag (Part BBa_J435329). We used AF_lacZ pDest cloning vector to build our construct.
Cycle 3: Build
To design the four antibodies required for our project, we BrioBricked four inserts as a composite part. Each composite part has a strong T7 inducible promoter, enabling IPTG induction, an E. coli codon-optimized ribosomal binding site (RBS), the antibody coding sequence, and a terminator. The RBS and antibody coding sequence were designed to serve a different purpose for each antibody. Legal restriction enzymes were also placed between each subcomponent. Next, we decided to use pSB3K3 for our vector, a low-copy number plasmid, which is important to reduce the metabolic burden on our cells.1
Cycle 4: Test - Protein expression
Escherichia coli DH5α was transformed with our composite part for the expression of our AMPs. Expressed proteins were purified and confirmed by SDS PAGE.
Future plan
We plan to further test our engineered peptides for their ability to inhibit S. aureus. If our AMPs do not give valuable inhibition zone after testing on S. aureus, we will have to improve on our bioinformatics analysis to find more gene clusters responsible for modifying AMPs. We will also redesign our construct if time permits.