iGEM 2021 — Team UFlorida

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# Team:UFlorida/Notebook

# Notebook

Document the dates you worked on your project. This should be a detailed account of the work done each day for your project.

### What should this page have?

* Chronological notes of what your team is doing.
* Brief descriptions of daily important events.
* Pictures of your progress.
* Mention who participated in what task.

### Inspiration

You can see what others teams have done to organize their notes:

* [2018 Munich](https://2018.igem.org/Team%3AMunich/Notebook)
* [2019 Georgia State](https://2019.igem.org/Team%3AGeorgia_State/Notebook)
* [2019 Newcastle](https://2019.igem.org/Team%3ANewcastle/Notebook)
* [2020 IISER Pune India](https://2020.igem.org/Team%3AIISER-Pune-India/Notebook)
* [2020 Lund](https://2020.igem.org/Team%3ALund/Notebook)
* [2020 NOVA LxPortugal](https://2020.igem.org/Team%3ANOVA_LxPortugal/Notebook)
* [2020 RDFZ China](https://2020.igem.org/Team%3ARDFZ-China/NoteBook)

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# Team:UFlorida/Engineering

UF iGEM 2021

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* [Team](https://2021.igem.org/Team%3AUFlorida/Team)

  [Team](https://2021.igem.org/Team%3AUFlorida/Team)[Attributions](https://2021.igem.org/Team%3AUFlorida/Attributions)[Collaborations](https://2021.igem.org/Team%3AUFlorida/Collaborations)
* [Project](https://2021.igem.org/Team%3AUFlorida/Description)

  [Description](https://2021.igem.org/Team%3AUFlorida/Description)[Results](https://2021.igem.org/Team%3AUFlorida/Results)[Contribution](https://2021.igem.org/Team%3AUFlorida/Contribution)[Model](https://2021.igem.org/Team%3AUFlorida/Model)[Engineering](https://2021.igem.org/Team%3AUFlorida/Engineering)[Implementation](https://2021.igem.org/Team%3AUFlorida/Implementation)[Experiments](https://2021.igem.org/Team%3AUFlorida/Experiments)
* [Parts](https://2021.igem.org/Team%3AUFlorida/Parts)
* [Human Practices](https://2021.igem.org/Team%3AUFlorida/Human_Practices)

  [Integrated Human
  Practices](https://2021.igem.org/Team%3AUFlorida/Human_Practices)[Education & Communication](https://2021.igem.org/Team%3AUFlorida/Communication)[Partnership](https://2021.igem.org/Team%3AUFlorida/Partnership)[Excellence
  in Another Area](https://2021.igem.org/Team%3AUFlorida/Excellence)

# ENGINEERING

## Design

At UFLORIDA we became interested in the idea of having a project further researching alternative selection
markers by taking inspiration from previous teams like [Edinburgh 2012](https://2012.igem.org/Team%3AEdinburgh/Project/Non-antibiotic-Markers). Based on
previous research showing how sugars can act as evolutionary driving forces in certain types of bacteria, we
decided to test how sucrose, Ribitol, and D-Arabitol would react as selection markers to *Escherichia.coli* and
*Pseudomonas.putida*. Each of these sugars was built using a broad range toolbox for synthetic biology created by
Layla Schuster and Dr. Christopher Reisch here at UF. Check out their paper here in our reference section. It is
very helpful and provides great tools for synthetic biology.

We will go through the engineering design by showing how we correctly assembled one of our plasmids: pFLtR5 in
*Escherichia.coli*. You can check our lab notebook and results sections for more information about our experiments
and other constructs we built. Click [here for more
information](https://2021.igem.org/Team%3AUFlorida/Experiments). pfLtR5 means we are working with ribitol(R5), (f) is the name of our origin fragment which is
RSF1010, (Lt) is for the regulator region tetR, and (p) means plasmid. We are also using gentamicin as our
bacterial marker to prove our target bacteria took our plasmid. See figure 1, 2, and 3 for the types of plasmids we
worked with and designed through benchling. We utilized the gene regulatory power of the lac and tet gene
networks to use them for controlled gene expression of our system. See figure 1 for a better explanation of it.
In the pFLtR5 circuit we are using the pLacI promoter to express the TetR regulator. This promoter natively
codes for the lacI repressor that regulates the lac operon. This is a strong promoter expressed constitutively
with regulation.The expression of TetR inhibits the gene activation of the downstream pTetO promoter/operator
regions by binding to the operator site of this region and preventing RNA polymerase from performing its job.It
is important to not that there is always leakiness in the system and as such a low induction of expression
without inducing the system can still happen.

This circuit we set up allows us to control the gene regulation of our ribitol sugar pathway by activating it
using atc as an inducer. Atc is a chemical that binds toTetR and causes a conformational change in the structure
of this dimer protein, rendering it unable to bind to the ptet-Operator site. Once TetR is unbound from our
desired ribitol metabolism coding sequences ,we can increase their expression as we add more atc. For this
specific pathway we need two genes: Ribitol dehydrogenase and Ribulokinase. These two genes are known to be used
for the breakdown of ribitol. The name dehydrogenase indicates that ribitol is an enzyme that causes an
oxidation reaction alongside a cofactor (Nad+), creating D-ribulose. We then use ribulokinase with the help of
ATP as an energy catalyst to transfer a phosphate group making D-ribulose phosphate. This product then further
goes on to be further broken down. By using the tools from the plasmid tool box paper, we have been able to
design a synthetic gene circuit using heterologous gene combinations attempting to further research alternative
selection markers. Next step is building our circuit through gibson assembly in situ.

![](https://static.igem.org/mediawiki/2021/a/aa/T--UFlorida--WS-basic--img--photos--engineering1.png)

Figure 1: Plasmid Nomenclature

![](https://static.igem.org/mediawiki/2021/f/f4/T--UFlorida--WS-basic--img--photos--engineering_ribitol.png)

Figure 2: Plasmid picture of Ribitol plasmid

![](https://static.igem.org/mediawiki/2021/4/4b/T--UFlorida--WS-basic--img--photos--engineering_pfciri.png)

Figure 3: Plasmid Benchling design of pFCiR5

## Build

Once we had our design with the desired genetic circuit for our carbon sugars like ribitol, our next step was
to order the individual parts of our plasmid with their respective primers and start assembling it.

What are primers? Primers are short sequences of nucleic acids like DNA that bind to complementary regions. They
are used so that the replication machinery of DNA can properly amplify a desired DNA sequence. They also can
serve a purpose in genetic engineering when it comes to assembling all of the individual parts of our plasmids:
RSF100 origin/backbone, Gent, Ribitol CDS, and Lter Regulator. Once we had all of the reagents needed, we used
PCR to amplify each of those sequences for a 50 microliter reaction in a PCR tube. This allowed us to have
enough stock DNA concentration to further proceed into the assembly of all of the parts at once. Colony PCR was
performed over 35 cycles with celsius temperatures of : 98 Denaturation, 60 annealing, and 72 elongation and
final extension. We used the super fi 2 DNA polymerase for the amplification of each part. For each PCR reaction
of our plasmid, we used DPN1 protein in order to get rid of the wild type DNA sequences that would not make part
of the circuit. The amplification of our parts was used with the primers mentioned in table 1.

| **Primer of pFLtR5** | **Number of primers** | **Size of pFLtR5 by part** |
| --- | --- | --- |
| RSF1010 | 2283,2818 | 6 kbs |
| Ltet Regulator | 2819,2329 | 1.3kbs |
| Gent Marker | 2282,1529 | 1kbs |
| Ribitol pathway | 2844,2845 | 2.5kbs |

Table 1: Size of included parts in pFLtR5

After amplifying our parts, we proceeded to assemble our plasmid using the method of gibson cloning.
This method relies on using overlapping primers of our parts that are complementary to each other, serving as
glue to assemble our parts together. It works by first using an exonuclease enzyme that degrades the desired
sequences in the 3 to 5 exonuclease direction. This allows for the overlapping primer hangs to bind to each
other, acting as glue and fully assembling our genetic circuit. For this reaction, we used a total mixture of 2
microliters. The solution was placed into a PCR machine for 1 hour at 50 degrees temperature. See figure 4 for
more reference.

![](https://static.igem.org/mediawiki/2021/9/9f/T--UFlorida--WS-basic--img--photos--engineering4.png)

Figure 4: Primer location in Gibson Assembly. Marker for shown plasmid is gentamicin

| **Gibson pFLtR5 plasmid assembly** | **Solution size** |
| --- | --- |
| Gibson assembly mix | 1 ul |
| RSF1010 Backbone | 0.25 ul |
| Ribitol pathway | 0.25 ul |
| Ltet Regulator | 0.25 ul |
| Gent marker | 0.25 ul |

Table 2: Parts in pFLtR5 Gibson Assembly

After we had assembled our plasmid, our next step was to chemically prepare our target bacteria to uptake our
plasmid via the process of transformation. This process allows us to hijack our bacteria into taking our plasmid
by exposing them to electricity or heat shock. These two types of methods trigger our target bacteria's membrane
to become more susceptible to the uptake of plasmid DNA. This process is physically straining for bacteria so we
need to transfer it to a liquid vial with fresh LB media for a recovery period. This period can be 1-2 hours of
incubation time. See figure 5 for more reference.

![](https://static.igem.org/mediawiki/2021/4/4a/T--UFlorida--WS-basic--img--photos--engineering5.png)

Figure 5: DNA Transformation

In order to check whether our circuit was taken up by our bacteria we extracted the plasmid from our bacteria
and we performed a PCR reaction amplifying the Ribitol pathway with the correct primers. Our gel results show
that we were able to assemble our plasmid correctly as the size of the migration in our part matches the
expected size we had from our design. See figure 6 for more information. Our next steps are to test whether we can
utilize these alternative selection markers that can be induced through our regulatory pathway.

![](https://static.igem.org/mediawiki/2021/5/54/T--UFlorida--WS-basic--img--photos--engineering6.jpg)

## Test

Once we had proved our plasmid was assembled correctly, our next steps were to test both the effectiveness and
efficiency of alternative sugar selection. Initial experimentation appeared promising as transformed growth
occurred; however, its inconsistency even among sugars proved it ultimately inconclusive. In an effort to
establish that introduction of our plasmid allowed for selective growth, we attempted to control for numerous
factors that might have confounded our results.

### Growth Phase

There were several factors we considered that might have potentially inhibited the success of our sugar
selection. The first of these cases was the phase of the parent colony. Initially, whenever we subcultured from
the parent colony which we had already successfully transformed we would subculture and immediately add the
respective inducer for the Ltet and begin an experiment. We hypothesized the issue with this being that the
parent colony, having already grown, existed in a stationary growth phase where minimal further colony growth
may be observed and biochemical processes are stabilized to that required for continued survival. Given a new
metabolic enzyme to produce may have either been downregulated due to the importance of other factors necessary
towards survival and thus enzyme production occurring at a very slow rate or rather not enough necessary
cellular machinery already present to produce the enzyme due to high levels during growth being depleted. To
address this issue, we performed an experiment in which we took two subcultures from the same parent colony. One
subculture, our control, was immediately treated with the appropriate inducer; the other subculture was again
quickly subcultured to obtain a colony in exponential phase which was only then treated with the respective
inducer. While the stationary phase may have inhibited enzyme production, we hypothesized that the addition of
an inducer during an exponential phase of growth would allow for a greater availability of cellular resources
and machinery to be dedicated towards enzymatic production. Moreover, the exponential phase subculture had more
time to produce the desired sugar enzymes.

Bacterial growth may be sorted into four distinct phases: lag, exponential, stationary, and death. Prefacing the
growth of a colony is the initial lag phase; bacteria in this phase may be considered metabolically active but
do not actively participate in growth. Moreover, the synthesis of essential proteins for survival and eventual
growth is established in this phase prior to that of the next phase. Only during the exponential phase do
bacteria begin to replicate. This stage is where selection occurs. Only cells maintaining one of our engineered
plasmids capable of metabolizing the only energy source available (the respective sugar) would be able to grow.
Metabolic conditioning is highest during this phase as cellular processes are most needed to support growth. We
were able to isolate various other potential energy sources through the introduction of M9 media, a minimal
media type not rich with available sugars as energy sources to encourage colony growth. The environment's
carrying capacity identifies the transition from exponential to stationary phase. As colonies compete for scarce
sugar availability, further growth is stifled likewise by limits in energy supply. Bacterial population plateaus
as growth rate equates to the death rate. Furthermore, metabolic activity significantly lessens as survival is
prioritized over sustained growth. Finally, an exhausted nutrient supply marks the beginning of compounding
death. Minimal growth may be observed as lysed cells may supply nutrients that spur occasional growth sites;
however, without environmental nutrient availability, such cells will quickly cease to replicate and also die.

### Inducer Effects

Rather than the phase of growth when conducting an experiment, another variable we sought to regulate was
whether the concentration of the respective inducer influenced the amount of enzyme produced. There are two
important implications regarding the effect of the inducer on the regulator. With too little induced production
of the enzyme, the cell may not be able to produce enough of the enzyme to metabolize the respective sugar and
thus will die. However, too much of the enzyme produced might draw cellular resources and machinery otherwise
vital to growth and survival, likewise leading to cellular death. In order to account for both these
possibilities, we conducted an experiment varying our inducer concentration from that we regularly used to those
both lower and higher amounts. No colonies grew on the respective sugar regardless of inducer concentration.

![](https://static.igem.org/mediawiki/2021/0/05/T--UFlorida--WS-basic--img--photos--engineering7.png)

Figure 7: Method of inducer application to experimental plates

Inducible systems are integral to the systemized optimization of desired outcomes in synthetic biology. By
utilizing our method of toolbox plasmid assembly, we were able to tightly regulate the effects of our
transformed pathways into their respective native hosts. Regulating systems are oftentimes initiated by a
promoter composite consisting of molecular interactions to commence/inhibit transcription of the desired
gene(s). Of the noted toolbox, we opted to construct two plasmids of each respective tested sugar. Each sugars'
pair of plasmids would contain one retaining the Ltet02 and the other holding the CinR regulator. We chose these
two regulators of the toolbox specifically for their previously proven ability to induce respective lower and
higher levels of transcription whilst also maintaining established inducible promoter systems. As a result, with
each respective sugars' plasmids, we would be able to compare whether a lesser or greater production of desired
sugar enzymes would better promote selection.

The Ltet02 system is also termed the tetracycline (tc) expression system. TetR is the primary protein regulating
the system. Natively, tetR binds to the tetO operator and blocks downstream transcription. Upon tc binding,
however, tetR releases from the operator and thereby facilitates transcription. We were able to manipulate this
system via the tightly coordinated addition of anhydrotetracycline (aTc) inducer. A derivative of tc, aTc
maintains a high affinity for tetR that allows for its introduction to synthetically promote transcription.

The CinR system natively operates in a positive feedback loop manner. Transcription of cinR both stimulates
downstream gene cinI and production of the 3-hydroxy-7-*cis* tetradecenoyl)-l-homoserine lactone (OHC14) molecule.
OHC further stimulates cinI. Transcription of cinI results in the production of another molecule closely related
to OHC14 which completes the positive feedback loop of cinI stimulation. In this manner, addition of OHC further
induces the system. One important note of the CinR quorum-sensing regulation system is that it has been found to
be strongly growth-inhibitory; an OHC14 derivative is known to be responsible for premature entry to the
stationary phase. Through experimentation, we found Ltet02 to better facilitate growth in all sugars' plasmids.

### Enzymatic Production

Finally, as growth phase, inducer influence, and sugar concentration were proven to not be primarily responsible
for the lack of expected select growth, we decided to further verify enzymatic production. With evidence
supporting the accuracy of our new engineered genetic part and inability to observe the expected growth of
transformed cells, we instead looked to any potential issue in the process of enzyme production itself. We were
able to assess the production of the desired enzymes coded for by our plasmids through an assay analysis. In
opting to evaluate the presence of coded-for dehydrogenase enzymes, we were able to take advantage of the
NAD/NADH cofactor system commonly present in a variety of metabolic systems through various lineages. By lysing
varying amounts of our transformed cells and immediately screening for a change in the ratio of NAD:NADH present
over time, we were able to evaluate for the expected trend of greater transformed cellular lysate equating to
greater changes of the NAD:NADH ratio in the observed time interval. Nevertheless, we found that compared to a
control in which no cell lysate and thus no dehydrogenase enzyme present to hasten the NAD:NADH conversion, the
observed varying concentration did not prove any conclusive association that evidenced greater dehydrogenase
present in lysate resulting in greater changes in the cofactor over time.

Similar to antibiotic resistance referring to cells' ability to grow and survive despite an inhospitable
environment, transference of enzymatic pathways aimed to confer the same ability to cells that would otherwise
die in an inhospitable environment. In this case, supplying only sugar non-transformed cells would be unable to
utilize as the sole energy source created the selective pressure antibiotics currently do. Enzymatic pathways
enabling the metabolism of each respective sugar were extrapolated from their native hosts. A complication in
the transference of sugar metabolic pathways is the series of enzymes that encode for vital components of the
process. Firstly, the selection of pathways must be limited to those simple sugars that require minimal
enzymatic interaction to break into usable monosaccharide carbon sources.

As aforementioned, a previous team was able to successfully induce metabolic genes necessary for sucrose
degradation to *E. coli*. This year, we sought to broaden the range and application of this metabolic transfer.
D-Arabinitol and Ribitol are both sugar alcohols that belong to a certain class of polyols termed pentitols. The
usefulness in this method of sugar selection is a common metabolic system of digestion.

* 1. The respective polyol dehydrogenase oxidizes the sugar to its respective keto sugar (D-Xylulose and
  D-Ribulose, respectively).
* 2. The respective keto sugar is phosphorylated by the sugar's corresponding kinase.
* 3. The resulting compounds enter the non-oxidative branch of the pentose phosphate pathway.

Only after the pentose phosphate pathway may a cell effectively utilize the resultant compounds to fuel vital
processes. A necessary aspect of this degradation system to be considered is that while the genes encoding for
the NAD-dependent dehydrogenase and ATP-dependent kinase may be transferred, the presence of a phosphorylated
keto sugar does not ensure that the third step, common to most bacterial strains, will commence.

The pentose phosphate pathway is separated into two distinct stages. The first, oxidative stage represents the
oxidation of a sugar to its determined phosphorylated keto sugar. Our transformed plasmids represent this
initial step of the pathway; we only supplied the necessary coding for this oxidation to occur with sugars
alternative to typical glucose. During the initial step of this oxidation, however, cofactor NADP+ is protonated
to NADPH. A potential reason why our system did not ultimately work may have been this aspect of the pentose
phosphate pathway. The concentration of NADP+ determines whether the derived keto sugar will continue down the
pathway. Lack of available NADP+ results in the halting of the pathway and accumulation of the unusable keto
sugar metabolite within the cell. Its concentration gradient favors against the pathway to an extent that, in
eukaryotes, glucose surges trigger a cellular need for NADPH for biosynthesis reactions as insulin likewise
upregulates production of the respective dehydrogenase. Furthermore, the most important distinction between the
first and second steps of the pathway remains that the former is an irreversible process whereas the latter is
reversible dependent on cellular needs.

D-Xylulose-5-phosphate (D-Xylulose) and D-Ribose-5-phosphate (D-Ribose) are the primary keto sugars that may
participate in the pathway. Thus, while the Arabitinol plasmid formed a valid keto sugar, the Ribitol plasmid
likely required the addition of two additional enzymes to convert unusable D-Ribulose to usable D-Ribose. One
necessary consideration made in the determination of how these processes would function was the space available
on each plasmid. At a certain size threshold, plasmids no longer favor conjugation. In order to promote
horizontal gene transfer, we had to determine which genes we thought to be most integral to an even suboptimal
functioning of the sugars' metabolic breakdown. Nevertheless, we did not account for the several reactions
occurring in the second, reductive step in the pentose phosphate pathway.

* 1. Transketolase enzyme transfers two smaller carbon chains to D-Ribose-5-Phosphate to form two new
  pentose sugars, A and B
* 2. Transaldolase enzyme facilitates the transfer of a 3-Carbon chain from molecule A to B, resulting in
  two new molecules, C and D
* 3. Transketolase uses D-Xylulose in conjunction with molecule C to form another of each molecules B and D
* 4. Both molecules B and D may then be utilized in traditional glycolysis

This series of reactions for the second, reductive step of the pentose phosphate pathway presents several other
opportunities for our plasmids' malfunctioning. With metabolism of D-Arabitol resulting in only D-Xylulose since
glucose that would have provided D-Ribose was absent, neither of the compounds that may be used for eventual
glycolysis would have been formed. Moreover, metabolism of Ribitol, even if the necessary enzymes for its
conversion to D-Ribose were present, would have lacked the required D-Xylulose to most efficiently produce
functional energy sources for glycolysis. This may have led to an inadequate amount of energy to fuel cellular
growth processes and kept cells in a survival-first state in the lag phase. Another consideration in the pentose
phosphate pathway is the direction of the process and whether it favors the phosphorylated keto sugar reactants
or the glycolytic-available products. If a cell needs ATP, it will follow the process to form products then
usable for glycolysis; however, a cell needing NADPH for other reductive, anaerobic processes will favor the
reactants by recycling the products. Finally is the foremost issue we may have encountered. With
D-Ribose-5-phosphate being essential to nucleotide formation, the reverse reactions are greatly favored in this
second step of the pentose phosphate pathway. As our cells would have entered an exponential phase and needed to
rapidly replicate genetic material for continued growth, the system providing their sole source of energy would
have been greatly favoring the reverse reaction and forming more reactant. Any minimal usable glycolytic product
would have been converted back to unusable molecules for growth, leaving the cells to die without any source of
energy. Such is one of the primary complications we found of using metabolism as a selection marker; offering a
single energy source creates a dipole towards where that energy is used towards. In this case, the growth of the
exponential phase would have inhibited growth by killing cells as survival in the lag phase was yet sustained.

Metabolism of sucrose is less detailed. There exists more than five variations of how certain bacterial strains
have come to gain the ability to utilize sucrose as a viable carbon source. We decided to use the variation of
the pathway successfully exhibited by a previous iGEM team in *E. coli*. In this pathway, uptaken sucrose is
oxidized in the periplasmic space by the corresponding dehydrogenase (D-Glucoside-3-dehydrogenase) to form a
keto sucrose sugar. Hydrolysis in the cytoplasm by the corresponding sucrose hydrolase enzyme (3-keto sucrose
hydrolase) finally separates the sucrose keto sugar to its monosaccharide derivatives that may be used in both
cellular respiration and glycolysis. We found that the previous iGEM team found selective success in alternative
selection by sucrose. By the metabolic pathway of sucrose catabolism utilizing a dehydrogenase enzyme, the
hydrolase enzyme is also necessary. Nevertheless, by transferring only the dehydrogenase gene, it is known that
that the encoded enzyme similarly allows for utilization of available lactose as another usable carbon sugar.
With our experimental design ensuring no other carbon source (i.e. lactose) was present alongside sucrose, we
may infer that lactose could have been present in the prior team's investigation. Additionally, our utilization
of an auxotrophic strain of *E. coli* for ease of transformation of our plasmids may have either not correctly
produced the necessary enzymes or perhaps not in the right location for sucrose metabolism.

Although we were unable to demonstrate sugar introduction as an alternative selectable marker, we were able to
successfully engineer and introduce a novel plasmid with the code for transferable sugar-metabolizing pathways
to *Pseudomonas.putida* and *Escherichia.coli*. Numerous complications exist in the manifestation of full
transference of a metabolic pathway. Many metabolites serve as complex intermediates within several systemic
pathways that play integral roles in cell function. Introducing mechanisms to metabolize new substances likely
incur innumerable opportunities to confound a natural pathway integral to the survival of a cell. Even with
fully analyzing the biochemical interactions responsible for metagenomic-derived processes, there are always
more details to be yet discovered to truly uncover why our system was unable to materialize. Moreover, there
additionally could have been complications arising from the aspect of synthetic biology. As we commonly used
transformed cells of one sugar as the positive control in an experiment meant to test another sugar, it may be
that the respective inducer added to stimulate the included regulator might have potential crossover with common
promoters among the other control. Finally, an exhaustion of cellular resources necessary to construct the coded
enzymes may have had compounding effects on those available for other cell functions as well. Further testing is
needed to establish a basis of consistency in expected selection prior to systemic optimization and judging
efficacy as a potential replacement for alternative selectable markers to traditional antibiotics; however, we
considered numerous variables integral to and were able to successfully produce the genetic parts to enable the
eventual creation of such a system. We've also been able to assemble each of our genetic circuits, arranging our
desired selection marker's CDS sequences with synthetic biology regulatory parts. We hope this contribution can
also inspire and help future teams interested in expanding the work of alternative selection markers using sugar
carbon sources

## Learn

Into our designed plasmids, we sought to include various enzymatic pathways in order to achieve our end result.
Ultimately, this end result would be conferring to a given species of bacteria the ability to utilize a specific
carbon source that it was not previously able to metabolize. During our research prior to the beginning of our
experimental process, we determined which species of bacteria we would be moving forward with during
experimentation on the basis of their ability to metabolize certain common carbon sources. From this preliminary
research, we came to the conclusion that we would be working with two distinct species: *Pseudomonas putida* (P.
putida) and *Escherichia coli* (*E. coli*). We would ideally provide the *E. coli* bacteria with the ability to
metabolize ribitol and the *P. putida* the ability to metabolize D-arabitol and sucrose. As can be gathered from
the Test section, we have much to learn both from the selection of these specific sugars as well as in our
implementation of the plasmids to the systems.

In future iterations of this project or similar projects, it would be beneficial to conduct intensive research
into the mechanisms of each of the enzymes responsible for the metabolism of their respective sugars in order to
better understand how these systems could go about being manipulated. On the same note, garnering a more
well-rounded understanding of the function of the enzymes themselves and their structures may serve to also
facilitate a more effective manipulation of enzymes. This is all being said as we attempted to confer our
bacteria with these newfound proteins without fully harboring an explicit understanding of how they will
function within the system itself as a whole and how these enzymes will serve the bacteria. In addition, this
research would allow us to improve upon the latter parts of the experiment in which we sought to alter the
concentration of the sugars in the media upon which our bacteria were growing to determine whether or not this
would have an effect on the transformation's efficacy. We could make these improvements in that we could
recognize the relationship between the sugar substrate and their respective enzymes to assess how a change in
concentration would alter the activity of the enzyme via an increase or decrease of activity.

From our data, we also determined a need to conduct our plasmid transformations at a time that would result in
the greatest amount of plasmid uptake and implementation into the bacteria's genome. For future experiments, we
would time the transformations to be done at a time in the life cycle of a bacterial colony at which the
bacteria are most prone to uptake and at which the largest percentage of bacteria residing in the colony would
harbor this characteristic. Having had an understanding of the times at which a bacterial colony will possess
these properties may have augmented the efficiency of our transformations and resulted in more positive results
such as a higher percentage of colonies within a plate exhibiting the desired trait(s).

During our experimental process, some of the growth we witnessed on the plates exhibited morphologies atypical
of either *P. putida* or *E. coli*. Figures 8 and 9 display the contamination in question. This finding suggests
contamination and the growth of a bacterial species that was neither of the species being studied. Mitigating
contamination during experiments when working with bacteria can be a very difficult process, especially when
some of the steps are done outside of a fume hood or away from the Bunsen burner. This difficulty is only
amplified when considering that our bacteria are being grown on plates containing glucose, sucrose, D-arabitol,
and ribose, all of which are sugar sources that will be ideal for the proliferation of certain bacterial
species, particularly glucose, which can be metabolized by nearly every species of bacteria and eukaryote. Our
liquid cultures were occasionally conducted in LB agar in order to allow the bacteria to proliferate to a
certain point before being spread along with their respective sugar-containing plates; this media serves as a
nutrient source for almost all bacterial species and is a very likely source of contamination. In order to
manage contamination within these liquid cultures, steps involving the test tubes containing LB agar were done
in proximity to a Bunsen burner flame, and all tools going into this culture were sterilized as well as the
outer rim of the glass test tube. This being said, we still encountered contamination in multiple plates and
liquid cultures. In future experimental iterations, this could best be managed by keeping all experimental
processes within a fume hood while continuing the use of the Bunsen burner. Additionally, given that our
experiments were conducted in a laboratory space shared by other students and researchers of the microbiology
department of the University of Florida, it is extremely likely that shared spaces contained trace amounts of a
wide variety of bacterial species.

![](https://static.igem.org/mediawiki/2021/2/28/T--UFlorida--WS-basic--img--photos--engineering8.jpg)

Figure 8: This image displays a sucrose plate on which we spotted a serial dilution of a *P. putida* culture
transformed with the plasmid pFLtRi5. Contamination can be seen in the form of white dots outside of the spots
located along the bottom right edge of the plate.

![](https://static.igem.org/mediawiki/2021/f/fb/T--UFlorida--WS-basic--img--photos--engineering9.jpg)

Figure 9: This image displays a ribitol sugar plate onto which a serial dilution of a *P. putida* culture
transformed with the plasmid pFLtSc5. Contamination can be seen in the form of white dots outside of the spots
located along the bottom right edge of the plate.

Lastly, a large and immensely important element of our experimental process was the design of our 5 plasmids.
These manufactured plasmids were referred to by the following names: pFLtAb5, pFCiAb5, pFLtSc5, pFCiRi5, and
pFLtRi5. The thought process behind the design of these individual plasmids can be found in the Design section
of Engineering Sucess. In order to verify the correct assembly of our plasmids and to verify that they contained
those elements that we sought to include in their design, these plasmids were sent to a company for sequencing.
Upon receiving these plasmids, we found that while most of them contained the desired elements (ie. sequences
for the respective sugar degradation pathway, gentamicin resistance, and the regulators), they contained
extraneous elements that could not be identified. This can be seen, for example in Figure 2. This figure is a
Benchling file of the sequenced pFLtRi5 plasmid. There are many elements within this plasmid that had not been
intentionally placed there, but as can be seen on the sequencing file, they were included in the chemical makeup
of the plasmid. This being said, the process of plasmid assembly will need to be more effectively micromanaged
in the future in order to mitigate inclusions of extraneous genes that may have an undetermined (ie. negative or
positive) effect on the overall experiment. The inclusion of bases with an unidentified effect on the overall
translational process of the plasmid and on the results of the translation may have altered our experimental
results in deleterious ways.

[![](https://static.igem.org/mediawiki/2021/8/8b/HQ_page_logo.jpg)](https://2021.igem.org)

# Team:UFlorida/Experiments

UF iGEM 2021

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# EXPERIMENTS

## **Read about our experiments!**

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# Team:UFlorida/Results

UF iGEM 2021

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* [Parts](https://2021.igem.org/Team%3AUFlorida/Parts)
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# RESULTS

## Summary

We used existing methods of transforming bacterial cells with a given plasmid in order to confer bacteria with
the ability to grow on a carbon source atypical of that species. In order to later assess whether or not both
the transformation and the plasmid design itself were successful, we ran control experiments that verified the
lack of certain sugar dehydrogenase pathways in *E. Coli* or *P. Putida*. For these experiments and all experiments
moving forward, the carbon sources used in our analyses were glucose, sucrose, ribitol, and D-arabitol. Given
these initial experiments, we moved forward with isolating the individual pathways that allowed our bacteria to
grow on their carbon source of choice and purified these parts.

Isolation and purification of these pathways allowed us to then amplify the corresponding parts such that we
could combine them into a plasmid of our design. Using Gibson HiFi assembly enzymes, these plasmids were
generated before being transformed into the appropriate bacteria via heat shock methods. Following this,
sequencing was used in order to determine whether or not our design had been properly incorporated into the
bacteria’s plasmid.

Once the cells were verified to have been successfully transformed, we moved forward in our experiment to assess
the bacteria’s responses to plasmid uptake in terms of whether or not the necessary enzymes would be produced.
In order to do so, we cultured the various transformed bacteria on those carbon sources on which they were not
initially able to grow to determine the extent to which the plasmid conferred to these bacteria the ability to
produce previously not present sugar dehydrogenase proteins. To test these newfound characteristics, time of
transformation, the concentration of the respective inducers, and concentration of sugar in the media were
varied and the results were observed.

## Project Achievements

### Successful Results

Over the course of the experiment we were successfully able to:

* • Establish negative controls for both *E. Coli* and *P. Putida*
* • Amplify parts isolated from the bacteria (origin of replication, a regulator of enzyme production, antibiotic resistance, respective sugar pathway)
* • Use gel electrophoresis in order to verify the presence of the correct amplified parts (see Figure 13)
* • Produce these plasmids: pFLtAb5, pFCiAb5, pFLtSc5, pFCiRi5, pFLtRi5 (see Figure 14)
* • Verify the sequence of these plasmids using sequencing programs (as seen in the image above)
* • Incorporate the aforementioned plasmids into *P. Putida* or *E. Coli* using heat shock or electroshock
  transformation
* • Observe the growth of these transformed bacteria

![](https://static.igem.org/mediawiki/2021/2/24/T--UFlorida--WS-basic--img--photos--results--results1.png)

Figure 1 - Establishing growth of *P. Putida* provided known usable carbon source (glucose)

![](https://static.igem.org/mediawiki/2021/d/d0/T--UFlorida--WS-basic--img--photos--results--results2.png)

Figure 2 - Establishing growth of *E. Coli* provided known usable carbon source (glucose)

![](https://static.igem.org/mediawiki/2021/c/c6/T--UFlorida--WS-basic--img--photos--results--results3.png)

Figure 3 - Control establishing no growth of *P. Putida* provided no usable carbon source

![](https://static.igem.org/mediawiki/2021/c/c5/T--UFlorida--WS-basic--img--photos--results--results4.png)

Figure 4 - Control establishing no growth of *E. Coli* provided no usable carbon source

![](https://static.igem.org/mediawiki/2021/9/92/T--UFlorida--WS-basic--img--photos--results--results5.png)

Figure 5 - Establishing natural growth of *P. Putida* provided experimental carbon source (Arabitol)

![](https://static.igem.org/mediawiki/2021/f/f8/T--UFlorida--WS-basic--img--photos--results--results6.png)

Figure 6 - Establishing natural growth of *E. Coli* provided experimental carbon source (Arabitol)

![](https://static.igem.org/mediawiki/2021/2/28/T--UFlorida--WS-basic--img--photos--results--results7.png)

Figure 7 - Establishing natural growth of *P. Putida* provided experimental carbon source (Ribitol)

![](https://static.igem.org/mediawiki/2021/d/d7/T--UFlorida--WS-basic--img--photos--results--results8.png)

Figure 8 - Establishing natural growth of *E. Coli* provided experimental carbon source (Ribitol)

![](https://static.igem.org/mediawiki/2021/8/86/T--UFlorida--WS-basic--img--photos--results--results9.png)

Figure 9 - Establishing natural growth of *P. Putida* provided experimental carbon source (Sucrose)

![](https://static.igem.org/mediawiki/2021/c/c5/T--UFlorida--WS-basic--img--photos--results--results10.png)

Figure 10 - Establishing natural growth of *E. Coli* provided experimental carbon source (Sucrose)

|  | Glucose | Arabitol | Ribitol | No sugar | Sucrose |
| --- | --- | --- | --- | --- | --- |
| *E. Coli* | 18 × 107 colonies/mL | 30 × 107 colonies/mL | 30 × 107 colonies/mL | 30 × 107 colonies/mL | 20 × 107 colonies/mL |
| *P. Putida* | 29 × 107 colonies/mL | 31 × 107 colonies/mL | 50 × 107 colonies/mL | 27 × 107 colonies/mL | 48 × 107 colonies/mL |

Figure 11 - Table establishes colony counts for baseline growth on control and on each type of provided sugar.
Note: Although colony counts might be similar between glucose and other three experimental carbon sources,
colony morphology is much clearer in glucose than the minute pinpoints observed with other sugars.

![](https://static.igem.org/mediawiki/2021/3/3f/T--UFlorida--WS-basic--img--photos--results--results11.png)

Figure 12 - Electrophoresis gel depicting verification of ribitol part of pFLtRi5 plasmid. The first band
(first from left) is a ladder dye used to delineate size of parts in 0.5kb increments. The second band (second
from left) was segmented using a forward primer in front of the respective regulator part (Ltet02) and reverse
primer internal to ribitol part at 1.5kb. The third band (third from left) depicts the entire ribitol metabolic
pathway as amplified through PCR at 2.5kb.

![](https://static.igem.org/mediawiki/2021/b/b8/T--UFlorida--WS-basic--img--photos--results--results12.png)

Figure 13 - Example of contamination often encountered over the course of experimentation. Spotted dilutions of
various plasmids on their respective sugars demonstrated highly dense growth among the supposedly least
concentrated spots (bottom right of both plates at bottom of image). When each contamination site was struck
onto plates, colony morphology was similar and not that of expected *E. Coli* (plate at top/middle of image).

![](https://static.igem.org/mediawiki/2021/8/8a/T--UFlorida--WS-basic--img--photos--results--results13.png)

Figure 14 - The sequenced pFLtRi5 plasmid designed by UFlorida iGEM 2021

### Unsuccessful Results

Prior to optimizing transformation efficiency, it was necessary to establish solely transformed cells’
utilization of respective sugars to colonize. Initial experimentation appeared promising as transformed growth
occurred; however, its inconsistency even among sugars proved it ultimately inconclusive. In an effort to
establish that introduction of our plasmid allowed for selective growth, we attempted to control for numerous
factors that might have confounded our results.

There were several factors we considered that might have potentially inhibited the success of our sugar
selection. The first of these cases was the phase of the parent colony. Initially, whenever we subcultured from
the parent colony which we had already found and proven to have successfully taken up our sugar plasmid, we
would subculture and immediately add the respective inducer for either the Ltet or Cin regulator and begin an
experiment. We hypothesized the issue with this being that the parent colony, having already grown, existed in a
stationary growth phase where minimal further colony growth may be observed and biochemical processes are
stabilized to that required for continued survival. Given a new metabolic enzyme to produce may have either been
downregulated due to the importance of other factors necessary towards survival and thus enzyme production
occurring at a very slow rate or rather not enough necessary cellular machinery already present to produce the
enzyme due to high levels during growth being depleted. To address this issue, we performed an experiment in
which we took two subcultures from the same parent colony. One subculture, our control, was immediately treated
with the appropriate inducer; the other subculture was again quickly subcultured to obtain a colony in
exponential phase which was only then treated with the respective inducer. While the stationary phase may have
inhibited enzyme production, we hypothesized that the addition of an inducer during an exponential phase of
growth would allow for a greater availability of cellular resources and machinery to be dedicated towards
enzymatic production. Moreover, the exponential phase subculture had more time to produce the desired sugar
enzymes.

Rather than the phase of growth when conducting an experiment, another variable we sought to regulate was
whether the concentration of the respective inducer influenced the amount of enzyme produced. There are two
important implications regarding the effect of the inducer unto the regulator. With too little induced
production of the enzyme, the cell may not be able to produce enough of the enzyme to metabolize the respective
sugar and thus will die. However, too much of the enzyme produced might draw cellular resources and machinery
otherwise vital to growth and survival, likewise leading to cellular death. In order to account for both these
possibilities, we conducted an experiment varying our inducer concentration from that we regularly used to those
both lower and higher amounts. No colonies grew on the respective sugar regardless of inducer concentration.

With neither the growth phase nor inducer concentration ultimately being the cause for why our transformed
plasmids were yet unable to consistently grow on their respective sugars, we next turned towards the
concentration of the sugars. Because cells would only be able to survive if able to produce enough of the
respective enzymes to metabolize the provided sugar, the concentration present could have impacted cell
survival. Too little sugar might yield not enough of an energy source to power other integral cellular activity
and likewise limit growth potential whilst ample sugar should theoretically allow for much greater growth
potential than what had been observed with the concentration provided through previous experiments. In order to
test the significance of sugar concentration on our transformed cells’ survival, we set up a liquid culture
experiment. To test the effect of sugar concentration, a single one of our plasmids were considered at a time.
Within liquid media, five various sugar concentrations were suspended and then inoculated with a subculture. One
set of five liquid cultures contained transformed cells that retained the appropriate enzyme for the suspended
sugar as the other set of five liquid cultures were inoculated with transformed cells retaining a different
plasmid for another sugar not present. As such, we were able to use our successfully transformed cells to
evaluate survivability. Ultimately, however, equivalent growth was observed in both the positive and negative
controls at lower sugar concentrations and cell debris in both the positive and negative at higher
concentrations. A potential cause of this unexpected result at higher sugar concentrations could have been that
cellular resources were exhausted in producing the desired enzyme in the presence of greater energy supply that
other necessary cell functions were inhibited or downregulated per the result of the present enzymatic activity.

An important factor was discovered later in our lab work that resulted in our needing to repeat experiments to
retain validity in our results. When exploring potential factors that might be causing our transformed cells to
not display expected select growth, we discovered the supplementary strain of *E. Coli* used was an auxotroph. As
a result of the strain utilized having been engineered to better be able to take up and retain genetic material,
certain experimentally designed strains are known to necessitate something not needed in the wild type. In our
case, we discovered that it was necessary to supplement amino acid Leucine in experimental design in order for
the cell to perform a variety of vital functions. This alteration in design was incorporated into all subsequent
experiments in addition to asserting prior experimental results.

Finally, as growth phase, inducer influence, and sugar concentration were proven to not be primarily responsible
for the lack of expected select growth, we decided to further verify enzymatic production. With evidence
supporting the accuracy of our new engineered genetic part and inability to observe the expected growth of
transformed cells, we instead looked to any potential issue in the process of enzyme production itself. We were
able to assess the production of the desired enzymes coded for by our plasmids through an assay analysis. In
opting to evaluate the presence of coded-for dehydrogenase enzymes, we were able to take advantage of the
NAD/NADH cofactor system commonly present in a variety of metabolic systems through various lineages. By lysing
varying amounts of our transformed cells and immediately screening for a change in ratio of NAD:NADH present
over time, we were able to evaluate for the expected trend of greater transformed cellular lysate equating to
greater changes of the NAD:NADH ratio in the observed time interval. Nevertheless, we found that compared to a
control in which no cell lysate and thus no dehydrogenase enzyme present to hasten the NAD:NADH conversion, the
observed varying concentration did not prove any conclusive association that evidenced greater dehydrogenase
present in lysate resulting in greater changes in the cofactor over time.

Although we were unable to demonstrate sugar introduction as an alternative selectable marker, we were able to
successfully engineer and introduce a novel plasmid with the code for transferable sugar-metabolizing pathways
to *Pseudomonas.putida* and *Escherichia.coli*. Numerous complications exist in the manifestation of full
transference of a metabolic pathway. Many metabolites serve as complex intermediates within several systemic
pathways that play integral roles in cell function. Introducing mechanisms to metabolize new substances likely
incur innumerable opportunities to confound a natural pathway integral to the survival of a cell. Even with
fully analyzing the biochemical interactions responsible for metagenomic-derived processes, there are always
more details to be yet discovered to truly uncover why our system was unable to materialize. Moreover, there
additionally could have been complications arising from the aspect of synthetic biology. As we commonly used
transformed cells of one sugar as the positive control in an experiment meant to test another sugar, it may be
that the respective inducer added to stimulate the included regulator might have potential crossover with common
promoters among the other control. Finally, an exhaustion of cellular resources necessary to construct the coded
enzymes may have had compounding effects on those available for other cell functions as well. Further testing is
needed to establish a basis of consistency in expected selection prior to systemic optimization and judging
efficacy as a potential replacement for alternative selectable markers to traditional antibiotics; however, we
considered numerous variables integral to and were able to successfully produce the genetic parts to enable the
eventual creation of such a system.

### Design considerations for future teams interested in markers:

Team UFLORIDA understands the importance of collaboration and building upon previous work. We hope our project can provide the educational means to inspire and help future teams continue the synthetic biology aspects in alternative selection markers. We were able to work on the wet lab aspect of this project through the summer. We understand that science takes time and with this opportunity, we have provided a list of controls and additional work we think future teams should consider for further applications:

* 1. Set up additional controls: If your plasmid does not seem to work, see if you can order the fully assembled plasmid from a third party company and test it on your target bacteria. This will help you further narrow down whether there are issues with the reading frame of your circuit or any other error malfunctions that can arise from plasmid assembly.
* 2. Design a plasmid that has a localization signal peptide like the MalE(BBa\_K3114001) secretion tag. This is a 26 amino acid sequence that is fused to the N-region of a protein. This tag can target our desired CDS and export it via the Sec secretion pathway to the periplasmic space. The project could also test the viability of different tags as extra procedures using the igem repository signal peptides for recombinant protein expression in our bacteria. This can help rule out whether our proteins would work if they can be exported to the periplasmic space. It seems that our CDS proteins are aggregating in the cytosolic space and as such trying different signal peptides with our system can result in further troubleshooting.

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