UF iGEM 2021



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. 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. 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.

Figure 1: Plasmid Nomenclature

Figure 2: Plasmid picture of Ribitol plasmid

Figure 3: Plasmid Benchling design of pFCiR5


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.

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.

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.

Figure 5: 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.


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.

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


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.

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.

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.