Team:UFlorida/Engineering

Harmful algal blooms, such as “red tide” caused by Karena Brevis, have a direct negative environmental and economic impact in Florida. Wide-scale fish kills and the release of neurotoxins harm wildlife that inhabit the coastal waters and people who depend on the environment to run their businesses. Eutrophication also contributes to a negative environmental impact in the state of Florida, as it causes excess algal growth. In turn, the excess algae deplete oxygen and essential nutrients for other organisms in the habitat, leading to the loss of biodiversity. A main contributor to eutrophication and harmful algal blooms are fertilizer runoffs from the land, with a major ingredient in fertilizer being phosphorous.

IMAGINE

Team Florida strives to protect our waterways and wetland ecosystems from this devastation. The first step in solving the problem is detection, so Team UFlorida has taken on the challenge of creating a biosensor that can detect above-safe levels of phosphorus in samples of water. This biosensor would allow ecologists to make informed decisions about the impacted waterways. The biosensor would be integrated into E.coli and would serve to quantify the amount of phosphorus in a given body of water.

DESIGN

Team Florida saw the potential to engineer a naturally occurring system to produce a desired response. This is where Team Florida built upon their previous project to couple the PhoB-PhoR system with SCRIBE and a genetic inverter that would allow this system to respond at phosphorous concentrations above 300uM instead of below these conditions.

By adding our own genetic inverter, we have genetically engineered the PhoB-PhoR system to enable it to respond to high phosphorus levels. The PhoB-PhoR network has a histidine kinase sensor (PhoR) and a response regulator protein (PhoB). When E.coli cells are low in phosphate, these organisms activate gene expression of genes like PhoR which serve as a sensor protein that when stimulated by the lack of phosphate, catalyzes the activation of response regulator PhoB. As a positive response regulator, PhoB activates the gene expression of itself, alongside PhoR and other genes essential for the intake of (Pi) into E.coli. PhoB then goes on to directly active promoter PhoA.

Tetr encodes for a repressor protein that comes directly after the pPhoA promoter. Then, there is a pTet promoter after the Tetr gene. In low phosphate conditions, PhoB binds to activate pPhoA leading to the transcription of the Tetr repressor gene. The Tetr repressor gene is then translated into the Tetr protein, which binds and represses the pTet promoter; therefore, SCRIBE is not transcribed.

Because pTet is a constitutive promoter, when the biosensor is placed in high phosphate conditions, PhoBR is turned off and the pTet promoter remains active due to the absence of Tetr. Thus, the SCRIBE system is under control of the pTet promoter. SCRIBE is transcribed and a mutation results in the bacterial chromosome.

BUILD

Unfortunately, COVID-19 has prevented us access to a lab. Team Florida has used this to their advantage by simulating this genetically engineered system in MATLAB based off of parameters found in literature as the first part of this two part project. Part two in 2021 will be to actually implement this into a lab to create the biosensor.

Despite access to a lab, Team Florida has come up with a thorough experimental design.

Plasmid Containing SCRIBE

Kanamycin resistance gene
pTet: Inverting regulator; ptet promoter is repressed by TetR
Reverse transcriptase: converts the ssRNA into ssDNA
Beta subunit: Binds to the ends of the target sequence to prevent degradation of the ssDNA

SCRIBE (Synthetic Cellular Recorders Integrating Biological Events) is a tool that uses modified retrons that have been transformed into bacterial cells to produce single-stranded DNA in response to a stimulus. The ssDNA is incorporated into the genome using the replication machinery of the bacteria. This technique mutates the bacteria to have rifampicin resistance.

Coupled with the PhoBR circuit, the inducer for SCRIBE is phosphorous. RNA polymerase will transcribe rpoB, turning dsDNA into mRNA. Reverse transcriptase then converts ssRNA into ssDNA, and the beta recombinase protein binds to the ends of ssDNA to help incorporate the DNA into the bacterial chromosome. The cells now contain the mutation and will exhibit rifampicin resistance.

PhoBR Plasmid (Modified version of pACYC184)

Ampicillin resistance gene
PhoR gene--sensor histidine kinase, phosphorylates PhoB
PhoA promoter
TetR- Genetic inverter repressor protein

Repeat the following steps for the SCRIBE plasmid and the PhoR-PhoB plasmid

PCR
Gel electrophoresis → mini prep → Dpn1 (recognizes methylation sites from wild types)
Cloning CPEC (The parts for the PhoB-PhoR cloning will be synthesized)
Transformation and plating
Colony PCR

TEST

In order to see if our experiments were carried out correctly, the outcomes would need to be evaluated by computing the fraction of cells that gain the mutation per generation. First, the cells would need to be induced by phosphorus, and plated with and without rifampicin. This will give the total cell count, as well as the number of cells with rifampicin resistance. Dividing the number of rifampicin resistant with total number gives the fraction of cells that have the mutation.

LEARN

Testing our biosensor in the lab will allow us to make informed decisions about our next steps. Since we did not carry out these experiments this year, we designed our experiments to plan for unexpected results to maximize our outcomes. Last year, our transformations were unsuccessful when our plasmids contained too many sequences. In order to avoid an unsuccessful transformation in the future, we will be using two plasmids that represent our designed genetic circuit for simplicity and optimality in terms of genetic context. In order to make our plasmids more efficient, we’ve distributed the assembly of our different DNA parts. One plasmid has the PhoBR genes alongside pPhoA and TetR. We have also added this system with terminator sequences from the voigt lab. The other plasmid includes the genes encoding the SCRIBE system and promoter ptet.

IMPROVE

We know that lab conditions are not representative of a natural environment. In order to account for this, conditions in the lab can be slowly varied to be representative of environmental conditions. From these results, we can get a more accurate prediction of how this biosensor will work in the real world.