iGEM 2023 — Team ualberta


{% extends "layout.html" %}
{% block title %}Engineering Success{% endblock %}
{% block lead %}Design, Build, Test, and Learn.{% endblock %}
{% block page\_content %}

[1. Overview](#overview)
[2.
Bio-Engineering](#bio-engineering)

[2.1. Choosing
Fluorescent Protein Pairs](#choosing-fluorescent)
[2.2.
Characterization of Fluorescent Proteins](#characterization-fluorescent)
[2.3. Creation
and Purification of Antigen Construct](#creation-purification)
[2.4. Following
BioBrick Standards](#following-biobrick)
[3.
Tech-engineering](#tech-engineering)

## OVERVIEW

Throughout the project, we performed multiple engineering cycles on both the tech and lab front, with
obstacles constantly needing to be addressed. How does the fluorescence of our reporters look visually?
Could it be detected by our software? What would protein production look like in our host cells? Does our
current FRET/quenching approach work in vitro? These were just some of the problems we came across and
wanted to overcome. In doing so, this led to different iterations of our Fungalescence synthetic construct.

## BIO-ENGINEERING

### Choosing Fluorescent Protein Pairs

The first challenge we came across was what fluorescent proteins would be viable for use in our
Fungalescence sensor. To facilitate this decision, we spent time determining the best expression platform
for distinguishing between fluorescent proteins. We also worked on deciding the optimal fluorescent proteins
for interaction with one another via FRET interaction. For ease and efficiency, we decided to transform
various fluorescent proteins containing plasmids into *E. coli* DH5a to create more copies of our DNA.These plasmids were also transformed into *E. coli* Rosetta-Gami (DE3) and induced on arabinose-containing plates to promote protein expression while under the control of pBAD. The colonies formed on plates would be used for an initial analysis as we expected to observe coloured colonies. Ultimately, we secured eight pBAD plasmids
containing eight different fluorescent proteins, which we simply transformed into DH5a. After
transformation, we found that mScarlet, mNeonGreen, sfGFP, and mClover3 were suitable for our construct due
to their visual colouring from the protein expression levels and transformation efficiency. We examined
emission and absorption spectra to further narrow down potential candidates. Ultimately, we chose mScarlet
and mNeonGreen as they allowed for a suitable excitation with green light and the ability for FRET between
the pair. However, this engineering cycle left us with some new questions to consider. Protein expression
using arabinose induction was not at sufficient levels for us to feel confident to incorporate it into our
design. If we were able to obtain higher protein expression, it would allow us to see a better signal that
could be detected by our drone software.

### Characterization of Fluorescent Proteins

With mScarlet and mNeonGreen as the chosen candidates, we wanted to create a new expression platform to
achieve higher protein production and allow us to better characterize our fluorescent proteins. Fortunately,
our secondary primary investigator was able to donate a pET-22b(+) plasmid under the control of a T7
promoter. This allows for IPTG induction, which is much stronger than arabinose induction. Therefore, we
decided to put our mScarlet and mNeonGreen coding sequences under the expression of the T7 promoter to
hopefully see more protein expression. We PCR amplified both genes from their pBAD vectors and ligated them
into the pET backbone using Gibson Assembly. After transformation into *E. coli* Rosetta GAMI and induced on plates containing IPTG, we did not see a significant difference in terms of saturation of colour in comparison to the pBAD constructs. Nevertheless, we were also able to successfully excite both red and green colonies using green light and were able to detect their fluorescence. We originally planned to have our construct fluoresce green in the presence of Fusarium graminearum and fluoresce red in the absence of our fungal pathogen due to FRET. However, after characterizing the fluorescence, we were unsatisfied with the nature of our reporter signals. After discussions with our engineering team, they explained that the green fluorescence was hard to detect and the colour did not contrast well with the shades of browns and greens that are observed within crop fields. Fortunately, mScarlet’s red fluorescence provided an ideal reporter signal as the red colour has significant contrast with the colours of the field environment environment.

After the results we observed in the lab, we chose to use mScarlet as the reporter signal. FRET interactions
rely on the transmission of energy between donor and acceptor molecules. In the case of fluorescent
proteins, donors emit light energy which excites neighboring acceptors, causing subsequent fluorescent
emissions. mScarlet fluoresces between ~550-650 nm (orange-red), severely limits our options for FRET
acceptors since very few fluorescent-type proteins can be excited by this range of light. After reviewing
the literature and various databases, we were able to identify a chromoprotein, ShadowR, that can quench
mScarlet through a FRET interaction that would serve our purposes well. This is what inspired us on our next
engineering cycle in the project involving the assembly, transformation, and expression of our
Cameleon-Q-Sensor in *E. coli* Rosetta-Gami B(DE3) and Shuffle-T7.

![](https://static.igem.wiki/teams/4755/wiki/engineering-success/figure-1.png)

**Figure 1. A.** Transformation plate of pET-22b(+) with Cameleon-Q-Sensor construct
in Rosetta-Gami. Plated on LB + 100mg/mL Ampicillin agar. We speculate that the purple colour of the
colonies is due to ShadowR quenching mScarlet. **B, C, & D.** Streak plating from transformation of
pJUMP with URS+ShadowR construct in SHuffle-T7. Temperature growth characterization from left to right:
25°C, 30°C, and 37°C.

### Creation and Purification of Antigen Construct

Simultaneously, as we were characterizing our fluorescent proteins, we got to work trying to express a
wild-type antigen of *F. graminearum* to act as a potential target to determine a nanobody for binding.
For this experiment, we thought to express our protein in *E. coli* Rosetta-Gami (DE3) to allow for its
purification with an associated HisTag. We utilized our pET plasmid with the T7 promoter as we could induce protein expression with IPTG and had access to an empty version of the vector. To reduce the complexity of our antigen, Endo-1,4-beta
xylanase enzyme (UniProt accession I1RII8), we searched for the exposed alpha-helical motifs on the enzyme
and put it into our helix backbone that had N-terminal and C-terminal helix initiators to control the
folding of the construct. As well, this contained a His-tag to make purification easier. However, when
ordering the construct from IDT, there was a miscommunication on the plasmid the insert would arrive in,
resulting in the loss of a terminator for proper transcription due to a different backbone being present.
Therefore we could not directly transform our vector into our competent cells. The restriction sites we were
originally planning to use for cloning were removed, so we could not remedy the problem with a simple
restriction digest. To solve this, we developed a PCR strategy where we linearized our pET backbone and
generated overhangs compatible with our PCR’d insert and combined them together with Gibson cloning.

To test protein expression and our purification methods, we first set out to test two different
purification methods. The resulting samples would be subjected to a Bradford assay and run on a
polyacrylamide gel to determine if protein expression worked. The first method we used was buffer-based
lysis, which allows for good quantities if the protein is hydrophobic. The second was using the French
Press, which works well provided the protein is hydrophilic. After performing the lysis and a Bradford
assay, we found that the French press was a much more effective method compared to the buffer lysis. As
such, we decided to use the French press in any further protein purifications. However, the numbers we
obtained from 100 mL of culture were still not satisfactory, thus we increased our amount of culture to 1L
for future applications. While visualizing the gel, we initially used a TGX stain-free method, however, we
were unable to visualize any bands. We then transitioned to a more traditional Coomassie blue method.
Unfortunately, the band that was developed as a result was undefined and faint. The subsequent decision was
made to utilize silver staining as it is more sensitive. Using this method more significantly defined bands
were visible. As such, we were able to create parameters for future protein purifications that served us
well in subsequent experimentation.

![](https://static.igem.wiki/teams/4755/wiki/engineering-success/figure-2.jpg)

**Figure 2.** DS-PAGE of purified antigen construct using buffer lysis and French
press, visualized using TGX-Stain Free.

![](https://static.igem.wiki/teams/4755/wiki/engineering-success/figure-3.jpg)

**Figure 3.** SDS-PAGE of purified antigen construct using buffer lysis and French
press, visualized using Coomassie blue staining.

![](https://static.igem.wiki/teams/4755/wiki/engineering-success/figure-4.jpg)

**Figure 4.** SDS-PAGE of purified antigen construct using French press, visualized
using silver staining.

### Following BioBrick Standards

As we proceeded through characterization of our fluorescent proteins and the design of our sensor
construct, our generated parts did not adhere to iGEM Biobrick standards. This is because we used cloning
strategies that were immediately convenient for the progress of our project and did not require us to order
any specific parts at the time. As such, we decided to make these parts compatible for submission to the
parts registry and under the expression of a different promoter. After using the T7 promoter contained
within pET-22b(+) for the majority of our engineering process, we found that while the promoter yielded good
results, optimizing the amount of IPTG to add to induce higher protein expression without becoming toxic to
the cell was difficult, as we experienced when we purified our antigen from large volumes of culture.
Furthermore, inducible promoters, such as the T7 promoter, are advantageous when the goal is to
strategically regulate protein expression, whereas constitutive promoters are useful when the goal is to
produce proteins continuously without requiring an inducer. With this in mind, we decided to redesign our
expression system and put our parts under the expression of a constitutive promoter to express more proteins
than with the IPTG inducible T7 promoter. This was also more cost effective as we no longer had to
purchase/use IPTG to produce protein. We developed a PCR strategy that amplified the biobricks parts that we
ordered, while adding the required prefixes and suffixes to adhere to iGEM submission standards.

It was decided that the pJUMP 28-1A plasmid, provided in this year's distribution kit, would act as the
entry vector for all of our basic and composite parts. A few features of this plasmid proved quite useful,
namely, the sfGFP insert and strong flanking terminators. The first allowed us to perform “green-white”
screening of successful ligation reactions. Due to pJUMPs terminators, it was no longer necessary to
incorporate our own terminator parts, which sped up the assembly process.

Using restriction cloning, we digested our biobricks parts and the pJUMP entry vector and utilized T4 DNA
Ligase to insert the biobricks parts into the backbone. We were able to combine and characterize mScarlet
and ShadowR under the control of the J23101-B0034 (BBa\_K4755001) part as well as we were able to combine the
Cameleon linker and ShadowR. First, we attempted transforming the constructs into *E. coli*
Rosetta-Gami B(DE3) which yielded no colonies. We postulate this was due to the shared Kanamycin resistance
of Rosetta-Gami and our plasmid. Next we attempted to transform in *E. coli* SHuffle-T7 which yielded
excellent results, resulting in bright colonies in terms of saturation of colour in comparison to our
previous constructs. Another distinction between the previous pET-22b(+) constructs and the new pJUMP
biobricks constructs was the observation of distinct colour in liquid cultures, indicative of increased
protein production. Unfortunately, the competition was coming to an end as these cloning were being
completed and we were unable to combine the URS-mScarlet and Chameleon-ShadowR biobricks constructs to
create the entire fungalescence sensory construct, but this would be the natural direction for the
completion of this stage of the engineering cycle.

![](https://static.igem.wiki/teams/4755/wiki/engineering-success/figure-5.jpg)

**Figre 5.** Initial transformation of DH5ɑ with the pJUMP+URS+mScarlet ligation
reaction.Three white colonies were identified. Plated on LB+ 50mg/mL Kanamycin agar.

![](https://static.igem.wiki/teams/4755/wiki/engineering-success/figure-6.jpg)

**Figre 6.** Re-streaked red colony from transformation of *E. coli* SHuffle-T7
with Colony 3 extracted plasmid. Red colour of colonies indicative of mScarlet production and success of
cloning. Plated on LB+ 50mg/mL Kanamycin agar.

![](https://static.igem.wiki/teams/4755/wiki/engineering-success/figure-7.jpg)

**Figre 7.** 50mL LB liquid + 50 µL 50mg/mL Kanamycin media inoculated from
re-streaked colony 3 SHuffle-T7 transformed with pJUMP+URS+mScarlet construct (Figure 6.). Red colour of
liquid indicative of mScarlet production and success of cloning.

![](https://static.igem.wiki/teams/4755/wiki/engineering-success/figure-8.jpg)

**Figre 8.** Initial transformation of DH5ɑ with the pJUMP+URS+ShadowR ligation
reaction. Four white colonies were identified. Plated on LB+ 50mg/mL Kanamycin agar.

![](https://static.igem.wiki/teams/4755/wiki/engineering-success/figure-9.jpg)

**Figre 9.** Re-streaked white colony from transformation of *E. coli* SHuffle-T7
with Colony 2 extracted plasmid. Gray/purple colour of colonies indicative of ShadowR production and
success of cloning. Plated on LB + 50mg/mL Kanamycin agar.

![](https://static.igem.wiki/teams/4755/wiki/engineering-success/figure-10.jpg)

**Figre 10.** 5mL LB liquid + 5µL 50mg/mL Kanamycin media inoculated with re-streaked
colony 2 SHuffle-T7 transformed with pJUMP+URS+ShadowR construct (Figure 9.). Tubes were placed on a UV
transilluminator to illuminate culture. Gray colour of liquid indicative of ShadowR production and
success of cloning.

![](https://static.igem.wiki/teams/4755/wiki/engineering-success/figure-11.jpg)

**Figre 11.** Initial transformation of DH5ɑ with the pJUMP+Cameleon+ShadowR ligation
reaction. Ten purple colonies were chosen for colony PCR to verify inserts. Plated on LB+ 50mg/mL
Kanamycin agar.

## TECH-ENGINEERING

### Tech-engineering

On the tech team side, most of our work had involved trial and error. When it came to designing for 3D
printing, we made edits and tested them using our university resources. This approach worked well, as we
encountered no issues with the trial-and-error method. In fact, the trial-and-error process proved effective
because it provided us with direct results and a better understanding of the areas we needed to improve.
This approach paralleled our work in AI development as well.

Upon receiving data (sample images) from the lab team, we embarked on optimizing the image masking
techniques to meet our project's requirements. This optimization occurred several times throughout the
project: once for the color we needed to detect, once for improvements based on that color, once for
determining the appropriate picture-taking distance, and one final optimization for the ultimate quality
checks.

Another area where we experimented with new techniques was during our camera
selection phase. We initially attempted to work with a multispectral camera; however, we encountered
difficulties connecting it to our Raspberry Pi, which made us appreciate the simplicity we would have had if
we had opted to use a regular RGB GoPro-like camera.

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{% extends "layout.html" %}
{% block title %}Results{% endblock %}
{% block lead %}8 months in the making!{% endblock %}
{% block page\_content %}

[1. Overview](#Overview)
[2. Characterization of Fluorescent Proteins in pBAD and PET-22b(+) Expression Systems](#Characterization of Fluorescent Proteins)
[3. Gibson Assembly of Q-Sensor and Antigen Constructs in pET-22b(+)](#Gibson Assembly)
[4. Protein Purification and Identification of Q-Sensor and Antigen Constructs](#Protein Purification)
[5. Nanobody Library Screening](#Nanobody Library Screening)
[6. gBlocks cloning in pJUMP28-1A](#PJUMP)

### Overview

The 2023 UAlberta iGEM team systematically worked through five key experiments over the course of our project. The team began the project by characterising various fluorescent proteins within the pBAD and pET-22b(+) expression systems. This step aimed to identify the most suitable fluorescent protein for integration into the sensory construct known as Fungalescence. Following this, the team shifted their focus towards assembling two essential components of the sensory construct. The Q-Sensor and Antigen cloning and expression in the pET-22b(+) system were successfully executed, including the subsequent protein purification and identification processes. The team then directed their attention to using the purified antigen in the nanobody library screening. The objective of this experiment was to identify a binder for the antigen fragment, with the ultimate goal of incorporating this binder into the design of Fungalescence. As the competition deadline approached, the team concentrated their efforts on cloning the gBlocks Gene Fragments of the Q-Sensor synthetic construct. This step was crucial for adhering to iGEM/BioBrick standard.

### Characterization of Fluorescent Proteins in pBAD and PET-22b(+) Expression Systems

As our project ultimately relied on the recognition of a fluorescent protein, it was crucial to decide which fluorescent protein is most easily expressed and visible for detection using the engineering team's drone system. Our first experiment involved the transformation of eight fluorescent proteins (sfGFP, mTurquoise2, eYFP,mScarlet, mTopaz, mClover 3, mNeonGreen, and mCitrine) under the control of the pBAD expression vector into Rosetta-Gami (DE3) cells. The intention of this experiment was to determine whether Rosetta-Gami (DE3) cells, when induced with arabinose, would produce fluorescent proteins visible to the naked eye and thus a drone detection system. After the transformation of the expression vectors containing the fluorescent proteins was achieved, we obtained the following results:

![Library Screening](https://static.igem.wiki/teams/4755/wiki/result/untitled-design-3.png)

**Figure 1.***E. coli* Rosetta-Gami transformations with eight different pBAD+fluorescent protein expression vectors. The transformation was pelleted, and 100µL (left) and 200 µL (right) of the resuspended transformation were plated on LB+100µg/mL Amp+2% Arabinose plates, grown overnight at 37°C. Fluorescent proteins being expressed bottom from left to right: mClover3, mTurquoise2, sfGFP, mTopaz.

These results showed that mScarlet and mNeonGreen had the most distinct colour, pink and green respectively, when visualised in ambient light. These preliminary results suggested that mScarlet and mNeonGreen would both be good candidates for the fluorescent protein to be used in our sensory construct.

We also wanted to compare the difference between the pBAD expression platform and the pET-22b(+) expression platform. Both required the use of inducible promoters but we hypothesised that the pET-22b(+) expression platform with its strong T7 promoter would result in even more distinctively coloured colonies (1).

We subsequently used Gibson Assembly to amplify the mScarlet, mNeonGreen, eYFP, and sfGFP genes out of the pBAD expression system and into the pET-22b(+) expression platform. We repeated the same transformation protocol as we did with the pBAD vector, transforming competent Rosetta-Gami cells with the pET-22b(+) fluorescent protein assemblies and inducing protein production on a plated culture. After the transformation of the expression vectors containing the fluorescent proteins was achieved, we obtained the following results:

![Library Screening](https://static.igem.wiki/teams/4755/wiki/result/untitled-design-4.png)

**Figure 2. Gibson Assembly of mScarlet and EYFP in pET22B(+).**All cells were transformed into *E. coli* Rosetta-Gami B(DE3) and plated on LB agar with 100 ug/mL Ampicillin, and 0.2 mM IPTG. Left plate shows Gibson assembled mScarlet induced with IPTG. The right plate shows gibson cloned EYFP also induced with IPTG.

Our results of the expression of the fluorescent proteins under the control of pET-22b(+) were not as expected; the colour of the colonies and, thus, the fluorescence was much weaker than that of the fluorescent proteins when under the control of pBAD. We postulated that the diminished expression of the fluorescent proteins in the pET-22b(+) vector could result from multiple occurrences. The first is the non-optimized concentration of IPTG, the inducer for the T7 promoter. We made our plates by adding 1 mL of 0.1M IPTG stock to the LB+Agar media. The IPTG stock solution may not have been effectively mixed throughout the media and distributed throughout the plates; thus, the inducer could have been found in higher concentrations on some plates and lower concentrations on others. If the IPTG stock had been mixed homogeneously throughout the media, there was still the possibility that the concentration of IPTG was too low in the volume of media it was added to induce protein production by the cells. Future experiments should focus on testing and optimising the concentration of IPTG in plated media for the expression of fluorescent proteins in the pET-22b(+) system.

Although the expression of fluorescent proteins in the pET-22b(+) platform was not as strong as in the pBAD platform, we could still visualise a distinct pink/red colour in the cells expressing mScarlet. The observation of increased production of mScarlet in relative comparison to the other fluorescent proteins we tested in both the pET-22b(+) and pBAD expression platforms, along with input from our engineering team, led to the ultimate decision to incorporate mScarlet into our final sensory construct.

### Gibson Assembly of Q-Sensor and Antigen Constructs in pET-22b(+)

Ultimately our team was successful in assembling the pET-22b(+)\_Q-Sensor and pET-22b(+)\_Antigen constructs. Please refer to Week 10-Week 12 of the Lab notebook for review and analysis of the results of these experiments.

### Protein Purification and Identification of Q-Sensor and Antigen Constructs

To prepare for the nanobody library screening and to perform our first test of our synthetic sensor, we sought to overexpress our antigen and Q-sensor constructs into E. coli Rosetta GAMI. After a mixup with our construct ordering, we were able to successfully ligate our antigen and Q-sensor fragments into a pET22b+ backbone using PCR. We confirmed the sequences of our plasmid using sequencing and colony PCR. These plasmids were then transformed into *E. coli* Rosetta GAMI competent cells and successful transformants were used to inoculate cultures for protein expression. We were able to successfully induce and purify our antigen protein using French press lysis, Ni-NTA columns, and the associated His-tag on our protein, and visualised using silver staining. The purified antigen constructs were then used for the nanobody library screening.

Please refer to Week 13 - 17 of the Lab notebook for in depth results of the protein purification and identification of Q-Sensor and Antigen constructs

### Nanobody Library Screening

We wanted to identify novel binders against a *Fusarium graminearum* antigen fragment. To facilitate this, our primary supervisor Dr. David Stuart gave our team access to his Yeast-based Nanobody (NB) library. Prior to screening, this library has approximately 2.5×109 unique NB binders. With the right screening protocol, isolating binders against almost any target protein becomes relatively easy. Our screening protocol is meant to decrease library diversity by iteratively forcing it to retain increasingly strong binders for the target of interest while discarding the rest.

Figure 3 provides a simplified overview of how we can select for and against target-specific binders in this screening protocol. The microbeads are magnetic, meaning they will latch onto the Magnetic Assisted Cell Sorting (MACS) column. If the bead is bound to anything, it will also stick to the column. Therefore, when positive binders stick to your target, that entire cell will remain bound to the column. While magnetised, you can perform several wash steps through the column, thereby eluting any negative binders. As a result, positive binders, of various strengths, are retained and used in subsequent screening rounds. It is important to note that positive binders are those with an affinity to your desired target protein, while negative binders lack this affinity. In the context of this library, binders are yeast cells with surface displayed NB variants. For our screening we were looking for a positive binder with an affinity for the *Fusarium* antigen fragment.

![Library Screening](https://static.igem.wiki/teams/4755/wiki/result/figure-3.png)

**Figure 3. Library Screening Using MACS. A.**Some library members have the desired target affinity, allowing them to form the complex shown in. Once complexed, the NB is carried over to the next round of screening. **B.** Alternatively, most library members lack the desired target affinity and cannot form the required complex. Any unbound NB will be washed through the MACS column and removed from the next round of screening.

An important part to this process is something called stringency. Low stringency allows almost all positive binders to be retained. Medium stringency is more strict, only allowing moderately strong, or stronger, binders to be retained. High stringency prevents all but the strongest binders from being retained. Generally, each screening step is accompanied by an increase in stringency. We increased stringency by decreasing the concentration of purified antigen fragments added to each screening round. Essentially, this radically reduces the number of binding spots available, which gives preference to stronger and stronger binders.

After three rounds of library screening we streaked out the enriched library on ~15 plates and cultured until single colony isolates appeared. We then selected 64 single colony isolates, culturing them for 2 days in a large 96 well plate, (Figure 4). In the same well plate we cultured eight WT S. cerevisiae isolates and four Naive library isolates to act as our controls, Figure 4. Note that this process is outlined in Figure 5. After 2 days we transferred the samples to a 96 well costar plate. Experimental groups A, and C-H received equal concentrations of purified antigen and anti-his antibody tagged with the Alexa FLuor 488 dye, Figure 4. Experimental group B was given no antigen and no antibody, Figure 4. Fluorescence values were obtained and corrected relative to OD values for each well.

![Values](https://static.igem.wiki/teams/4755/wiki/result/figure-4.png)

**Figure 4. Corrected Fluorescence Values For Potential Antigen-Fragment Binders.** Columns 1 and 2, *S. cerevisiae* and Naive Library are used here as controls. The WT *S. cerevisiae* are not expressing NBs and should therefore lack any significant binding affinity to the antigen fragment. Similarly, the randomly chosen Naive library members should have no significant binding affinity to the antigen fragment. With these parameters we can identify those isolates that are most likely to bind our antigen fragment.

![Determination Assay](https://static.igem.wiki/teams/4755/wiki/result/figure-5.png)

**Figure 5. NB Binder Determination Assay.**Streak out the enriched NB library. Select as many single colony isolates as desired and culture them with a large 96 well plate for ~2 days. Transfer isolates to a 96 well costar plate. Mix your antigen and a fluorophore labelled antibody that binds to your antigen with the transferred isolates. Measure fluorescence intensity to identify potent binders. Note: the left circle depicts the loss of fluorescence due to fluorophore washout. This would indicate the particular isolate is a poor binder. The right circle depicts the opposite situation, indicating a strong binder.

After comparing corrected values for each experimental group we identified several wells with potential binders and proceeded to culture them in preparation for plasmid isolation. Unfortunately, PCR amplification and DNA sequencing proved impossible for all but one of the isolates, that being E-7 (experimental group E row 7). However, we were able to obtain the NB sequences from four E-7 colonies and generated a 3D model of the NB, Figure 6.

![3D Model](https://static.igem.wiki/teams/4755/wiki/result/figure-6.gif)

**Figure 6. E-7 NB Isolate 3D Model Generated In CollabFold.** CDR1 is shown in gold. CDR2 is shown in green. CDR3 is shown in blue. The remainder of the NB structure is shown in red. Note: that we will not be providing the sequence to this NB on our Wiki or on the parts page.

### gBlocks cloning in pJUMP28-1A

Please refer to Week 17-21 of the lab notebook for an in depth overview and analysis of the results of the gBlocks cloning in pJUMP28-1A.

#### Characterization of mScarlet and ShadowR

To characterise the growth and colour of our mScarlet and ShadowR composite parts (BBa\_K4755015 and BBa\_K4755017, respectively) we decided to culture transformed *E. coli* Shuffle-T7 cells in liquid cultures (LCs) and on LB agar plates (plates for short) at the various growth temperatures of 25°C, 30°C, and 37°C. We first re-streaked single colonies from the transformation plates. Interestingly, it took several attempts at re-streaking various colonies before we were able to obtain adequately colourful cells. From that handful of plates, we chose two from which to obtain single colonies for the characterization, shown in Figure 7 A-C.

Nine colonies were selected from each plate and re-streaked in triplicate on the experimental plates shown in Figure 7 D-G. LCs were not done in triplicate because neither plate had enough single colonies, which is why we had a single culture for each temperature. It is worth noting that mScarlet colonies continuously appeared alongside sfGFP colonies, despite several attempts at re-streaking. However, none of our ShadowR plates exhibited this behaviour. Further investigation is required to fully understand why these results occurred.

![ Day 0 of Part Characterization](https://static.igem.wiki/teams/4755/wiki/result/figure-7.png)

**Figure 7. Day 0 of Part Characterization (0 Hours of Growth). A, B, & C.**mScarlet and ShadowR plates used to obtain single colony isolates for part characterization. **D, E, F, & G.** LB agar plates with 50ug/ml Kanamycin. All plates either contain ShadowR (BBa\_K4755017) or mScarlet (BBa\_K4755015) transformed into E. coli Shuffle-T7. ShadowR was streaked onto the left-side plates, which will be cultured at, from top to bottom, 25°C (Room Temp / RT), 30°C, and 37°C. mScarlet was streaked onto the right-side plates, which will be cultured at, from top to bottom, 25°C, 30°C, and 37°C.

Plates and LCs were left at their respective temperatures for ~24 hours. In the case of the LCs, they were also shaking at 150-200 rpm. Note that the controls displayed in Figure 8 H were placed with their respective experimental group for the duration of experiment. After ~24 hours, the plates and LCs were imaged with and without UV light exposure, (Figure 8). We do not possess the necessary equipment to properly visualise mScarlet at or around its maximum emission wavelength. However, while identifying optimal donor plates for the experiment, we decided to estimate sfGFP contamination by imaging each plate on a UV transilluminator. Despite its poor fluorescent response at this wavelength, mScarlet colonies appeared bright, having great contrast with sfGFP colonies. Therefore, we decided to determine the extent of mScarlet expression by visualising experimental groups with and without a UV transilluminator, Figure 8. For consistency, ShadowR plates and LCs were imaged in the same way.

ShadowR appears to have robust growth at all three temperatures, albeit with a noticeable decrease in cell density at 25°C, Figure 8. After ~24 hours, mScarlet shows the most growth and colour production at 37°C. Curiously, cell density appears to differ significantly between mScarlet plates and LCs. Because mScarlet had stunted growth at 25°C and 30°C, we would expect a similar drop in LC cell density for those same temperatures, which is not the case. To prevent further colony overgrowth we placed the 37°C mScarlet plate, 37°C ShadowR plate, and the 30°C ShadowR plate at 4°C. The 25°C mScarlet LC had no apparent growth after the 24 hours time point. To ensure that the LC was actually inoculated, we cultured the sample at 37°C. All remaining experimental groups were left to culture at their respective temperatures for another 24 hours.

![ Day 1 of Part Characterization](https://static.igem.wiki/teams/4755/wiki/result/figure-8.png)

**Figure 8. Day 1 of Part Characterization (~24 Hours of Growth).**LB agar plates and LB broth (5ml) with 50ug/ml Kanamycin. All plates and tubes either contain ShadowR (BBa\_K4755017) or mScarlet (BBa\_K4755015) transformed into *E. coli* Shuffle-T7. **A.**mScarlet (left) and ShadowR (right) grown at 37°C for ~24 hours. Imaged without UV exposure. **B.**mScarlet (left) and ShadowR (right) grown at 30°C for ~24 hours. Imaged without UV exposure.**C.**mScarlet (left) and ShadowR (right) grown at 25°C for ~24 hours. Imaged without UV exposure. **D.** From left to right, ShadowR grown at 37°C, 30°C, and 25°C for ~24 hours. Imaged with a UV transilluminator. **E.** From left to right, mScarlet grown at 37°C, 30°C, and 25°C for ~24 hours. Imaged with a UV transilluminator. **F.** Image labels indicate construct and culture conditions. Tubes were grown for ~24 hours and imaged using a UV transilluminator. **G.** Image labels indicate construct and culture conditions. Tubes were grown for ~24 hours and imaged without UV exposure. **H.** Negative controls containing 50ug/ml Kanamycin, prepared in an identical fashion to all other tubes and cultured at all three temperatures for ~24 hours. Note: While obtaining image F, we used a camera flash which seems to have darkened the photo.

After ~48 hours of growth, all groups were imaged as in previous time points. Note that the 4°C mScarlet and ShadowR plates were taken out of cold storage and placed alongside the other groups for imaging. The mScarlet and ShadowR LCs of Figure 9 A have a noticeable colour difference, with mScarlet being a slightly darker orange than the ShadowR samples. The difference is exacerbated when seen with a UV transilluminator, Figure 9 B, which causes all three mScarlet LCs to appear as a yellow-orange colour, while the ShadowR LCs are substantially darker.

Interestingly, the 25°C mScarlet LC that was moved to 37°C for 24 hours displayed a particularly deep orange hue, indicative of mScarlet, Figure 9 B. More importantly, after ~48 hours, the 37°C and 30°C mScarlet LCs show colour production, Figure 9 A. However, when imaged with the UV transilluminator, the 37°C mScarlet LC takes on a prominent yellow-green glow; probably indicated substantial sfGFP contamination, Figure 9 B. To a lesser extent, this is also true for the 30°C mScarlet LC, Figure 9 B. Contamination with sfGFP may explain the cell density discrepancy mentioned above.

After ~48 hours of growth at 25°C, the mScarlet plate remained mostly barren, Figure 9 C and D. The additional 24 hours only seems to have allowed mScarlet producing cells on the 30°C mScarlet plate to mature, Figure 9 C and D. In contrast, the 25°C ShadowR plate had a significant improvement in one of its three quadrants, Figure 9 E and F. ShadowR plates over an active UV transilluminator in Figure 9 F have a slight blue glow. This most likely arose due to a combination of factors present during the time of imaging. It is possible that ShadowR has a yet undescribed capacity for fluorescent photoconversion. However, no such traits were reported in its original publication or any previous publications relating to its precursor chromoprotein Ultramarine (2-4).

![ Day 2 of Part Characterization](https://static.igem.wiki/teams/4755/wiki/result/figure-9.png)

**Figure 9. Day 2 of Part Characterization (~48 Hours of Growth).**LB agar plates and LB broth (5ml) with 50ug/ml Kanamycin. All plates and tubes either contain ShadowR (BBa\_K4755017) or mScarlet (BBa\_K4755015) transformed into E. coli Shuffle-T7. **A.** Image labels indicate construct and culture conditions. Tubes were grown for ~48 hours and imaged without UV exposure. **B.** Image labels indicate construct and culture conditions. Tubes were grown for ~48 hours and imaged using a UV transilluminator. A separate control was prepared (without Kanamycin) and imaged alongside the other tubes. **C.** From left to right, mScarlet is grown at 25°C, 30°C, and 37°C for ~48 hours. Imaged without UV exposure. **D.** From left to right, mScarlet is grown at 25°C, 30°C, and 37°C for ~48 hours. Imaged with a UV transilluminator. **E.** From left to right, ShadowR grown at 25°C, 30°C, and 37°C for ~48 hours. Imaged without UV exposure. **F.** From left to right, ShadowR grown at 25°C, 30°C, and 37°C for ~48 hours. Imaged with a UV transilluminator.

Because mScarlet LCs began producing colour after ~48 hours, we decided to inoculate a much larger volume (50ml) with what we hoped was a pure mScarlet colony from the plate in Figure 7 A. After ~48 hours at 37°C and 200 rpm, we were delighted to see a deep orange colour in the flask, Figure 10 A & B. The only difference between the top row of Figure 10 and the bottom is culture duration. Images in the top row were taken ~48 hours after inoculation. Images in the bottom row were taken 96 hours after inoculation.

![ Day 2 of Part Characterization](https://static.igem.wiki/teams/4755/wiki/result/figure-10.png)

**Figure 10. Day 3 & 4 of Part Characterization (~48 & 96 Hours of Growth).**LB broth (50ml) with 50ug/ml Kanamycin. Culture was inoculated with mScarlet (BBa\_K4755015) transformed into *E. coli* Shuffle-T7 isolated from the left plate in Figure 7 C. **A.** The mScarlet isolate was grown in LB broth for ~48 hours. Imaged without UV exposure on October 2nd, 2023. **B.** The mScarlet isolate was grown in LB broth for ~48 hours. Imaged with a UV transilluminator. October 2nd, 2023. **C.** The mScarlet isolate was grown in LB broth for ~96 hours. Imaged without UV exposure on October 4th, 2023. **D.** The mScarlet isolate was grown in LB broth for ~96 hours. Imaged with a UV transilluminator. October 4th, 2023.

In conclusion, when cloned into the high copy pJUMP28-1A backbone, our ShadowR composite part (BBa\_K4755017) occasionally produces a faint purple colour. Regardless, our results strongly suggest that this construct can be efficiently cultured at 30°C or 37°C for ~24 hours when grown on LB agar plates or in liquid cultures. When cloned into the high copy pJUMP28-1A backbone, our mScarlet composite part (BBa\_K4755015) emits a vibrant orange coloured light when grown on LB agar plates or in liquid cultures. Our results strongly suggest that this construct should be cultured at 37°C for ~24 hours, when grown on LB agar plates, and ~48 hours, when grown in liquid cultures.

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## REFERENCES

1. Balzer, S., Kucharova, V., Megerle, J., Lale, R., Brautaset, T., & Valla, S. (2013). A comparative analysis of the properties of regulated promoter systems commonly used for recombinant gene expression in escherichia coli. *Microbial Cell Factories*, 12(1).<https://doi.org/10.1186/1475-2859-12-26>
2. Li, Y., Forbrich, A., Wu, J., Shao, P., Campbell, R. E., & Zemp, R. (2016). Engineering Dark Chromoprotein Reporters for Photoacoustic Microscopy and FRET Imaging. *Scientific Reports*, 6(1). <https://doi.org/10.1038/srep22129>
3. Murakoshi, H., Horiuchi, H., Kosugi, T., Onda, M., Sato, A., Koga, N., & Nabekura, J. (2019). ShadowR: a novel chromoprotein with reduced non-specific binding and improved expression in living cells. *Scientific Reports*, 9(1). <https://doi.org/10.1038/s41598-019-48604-4>
4. Pettikiriarachchi, A., Gong, L., Perugini, M. A., Devenish, R. J., & Prescott, M. (2012). Ultramarine, a Chromoprotein Acceptor for Förster Resonance Energy Transfer. *PLoS ONE*, 7(7). <https://doi.org/10.1371/journal.pone.0041028>

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