Difference between revisions of "Team:Paris Bettencourt/Proof Of Concept"

Introduction

In imagining a future where Staphylococcus epidermidis could be used as a vector for topical therapeutics, we knew that we needed a proof of concept that our epiFlex parts were functional. Thus, epiGlow was born!

We opted to optimise a transformation protocol for the expression of fluorescent proteins in S. epidermidis. This is a classic reporter system for genetic experiments and an easy assay for measurement. Additionally, this would allow for a streamlined characterization of different combinations of our regulatory sequences, such as promoter, RBS and terminator combinations.

Outside of our project, the ability to visualise staphyloccoci is an important resource, such as being able explore their behaviour in their native contexts such as their niche in the skin microbiome. Ofcourse this is ultimately a stepping stone for more ambitous pursuits such as the expression of genetic circuits for microbial population control or secretion of therapeutic proteins.

To realize our vision of fluorescent S. epidermidis, there were two main factors to consider: preparing DNA appropriately such that S. epidermidis would take it up and express it, and the actual physical transformation thereof. In DNA preparation, we could ensure more optimal expression of coding sequences through codon optimisation of our parts as the S. epidermidis genome has a relatively low GC content (1). The more pressing challenges included evading the restriction barriers in S. epidermidis and making it competent.

Restriction Barriers in Staphylococci

Part of the challenge in introducing recombinant DNA in staphylococci is getting past their restriction mediated modification systems. This is part of their immune system to protect against bacteriophages. Essentially, the methylation pattern of exogenous DNA is recognised as foreign and results in it being degraded by this system. Thus, any attempt to introduce our constructs require us to ensure that the methylation of the plasmid won't result in its degradation. To this end we were fortunate to be in contact with Dr. Ian Monk, who has worked on transforming staphylococci for years and gave us a few pointers.

Many Staphylococci, have different methods for DNA methylation, so in cloning experiments it becomes necessary to pass plasmid preparations through a strain of E. coli that can reproduce the methylation pattern, thus evading the restriciton barrier. Alternatively groups have passed DNA through an S. aureus strain (RN4220) that lacks a restriciton barrier, before transforming into their target strain(2). Universally, DNA-cytosine methyltransferase activity in E. coli needs to be eliminated to prepare DNA for all staphylococci, and luckily for S. epidermidis, a dcm-E. coli strain is sufficient for cloning purposes(3). In our pipeline we used a commercial dam-/dcm- strain which was not very efficient for cloning, but good for storage, but in future E. coliDC10B is suitable for both cloning and storage of constructs to express in S. epidermidis(4).

Transforming S. epidermidis

Much like E. coli, S. epidermidis is not naturally competent therefore transforming them is not a trivial task. Despite early attempts to introduce chemical competency to them, electroproation remains the gold standard. Despite this, transformation efficiency remains a problem, which was pointed out to us by leading skin microbiome researcher, Dr. Julia Oh.

Within our project we explored optimised electroporation protocols(3) and a newer heatshock/electroporation combination(5), to see which protocol would yield more transformants.

Protocols

For our workflow, we needed to be able to transform atleast 5µg of plasmid DNA lacking Dcm methylation per transformation. As we did not have E. coliDC10B at our disposal, what would be a 2-step cloning and transformation protocol was a 3-step protocol as we had to clone into an an efficient cloning strain before growing our plasmid in our dam-/dcm- E. coli and then purifying from there to electroporate into S. epidermidis.In our electroporation steps we explored a protocol from a recent preprint which only required the use of 1µg of DNA, the protocols for the aforementioned experiments can be found at the links below:

Plasmid Preparation

Electroporation Protocol

Electroporation + heatshock Protocol

Figure 1: Diagram of workflow from plasmid preparation to electroporation of S. epidermidis. Our three step pipeline requires an initial transformation of the epiFlex Golden Gate reaction into E. coli TOP10, followed by a purification of the extracted plasmid into dam-/dcm- E. coli from which the unmethylated construct can be purified and electroporated into S. epidermidis.

Results

Here we present our latest data on this ongoing mission to produce fluorescent S. epidermidis. For our proof of concept, we assembled a P1 epiFlex construct containing the SarA_P1 promoter, SodA RBS, mCherry codon optimised for expression in S. epidermidis, and the biobrick T7TE-LuxIA terminator. Our P1 backbone has kanamycin selection for E. coli and erythromycin selection for S. epidermidis.

Figure 3: Diagram of transcriptional unit assembled in the epiFlex p1 bacbone.

Figure 3: Graph of attempted measurement of mCherry fluorescence intensity in S. epidermidis and E. coli with or without epiFlex plasmid PBEF1_mCherry_AB. Graph depicts one independent experiment performed in triplicate.

Figure 4: Graph of OD600 measurements of S. epidermidis and E. coli over 20 hours with or without epiFlex plasmid PBEF1_mCherry_AB. Graph depicts one independent experiment performed in triplicate.

Figure 5: Imaging of plated bacteria with or without epiFlex mCherry construct (PBEF1_mCherry_AB). Image is a result of excitation and emission of plates according to mCherry spectral specifications.

Discussion

Using our epiFlex system we were able to successfully build a construct that expressed mCherry, we saw evidence of this on our E. coli growth plates before transforming into S. epidermidis(evident by the pink colonies), and saw the same in S. epidermidis.

In testing the growth dynamics of our transformed bacteria, we saw a slight decrease in the growth rate due to the presence of our construct in both S. epidermidis and E. coli, this is most likely due to an increased metabolic burden from expressing the heterologous gene product,although this remains to be experimentally determined.

We simultaneously attempted to measure mCherry fluorescence over time but were unable to optimise the measurement parameters in time.

Instead, we plated E. coli and S. epidermidis in parallel and imaged the fluorescence. As seen in figure 5, we successfully expressed mCherry in both S. epidermidis and E. coli,although through visual inspection we found the level of expression to be greater in E. coli than S. epidermidis.

Further work will go into improving transformation efficiency from electroporation and continuing to make constructs for the expression of other fluorescent proteins from the epiFlex toolbox. Additional focus on inducible expression modules will also bring us a step further into realizing the vision for a skin micriome chassis for synthetic biology.

References

• 1. Zhang, Yue‐Qing, et al. "Genome‐based analysis of virulence genes in a non‐biofilm‐forming Staphylococcus epidermidis strain (ATCC 12228)."Molecular microbiology49.6 (2003): 1577-1593.
• 2. Schenk, Steve, and Richard A. Laddaga. "Improved method for electroporation of Staphylococcus aureus."FEMS Microbiology Letters94.1-2 (1992): 133-138.
• 3. Lee, Jean YH, et al. "Mining the Methylome Reveals Extensive Diversity in Staphylococcus epidermidis Restriction Modification."Mbio10.6 (2019).
• 4. Monk, Ian R., et al. "Transforming the untransformable: application of direct transformation to manipulate genetically Staphylococcus aureus and Staphylococcus epidermidis."MBio3.2 (2012).
• 5. Chen, Y. Erin, et al. "Decoding commensal-host communication through genetic engineering of Staphylococcus epidermidis."bioRxiv(2019): 664656.