iGEM 2022 — Team ubc-okanagan

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[JUNE](#june)
[JULY](#july)
[AUGUST](#august)
[SEPTEMBER](#september)
[OCTOBER](#october)
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**Abbreviations:
*Synecho.* = Caffeic acid producing *Synechocystis* sp. PCC 6803, graciously donated by Laura
Furelos Brey and her team at the University of Copenhagen.
*‘Synecho.’* = Refers to what we originally thought was *Synechocystis* sp. PCC 6803 Glucose
Tolerant Stain (GT-S).
To read how we discovered we were not culturing glucose-tolerant *Synechocystis*, see weeks
August 28-September 3 and September 4-September 10.**

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# June

# July

# August

# September

# October

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[INTRODUCTION](#intro)
[DESIGN](#design)
[BUILDING](#building)
[TESTING](#testing)
[LEARNING](#learning)
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**Inspired by the natural wonder of bioluminescence, our team applied the engineering principles of the iGEM
Engineering Cycle to the development of an autobioluminescent system. The engineering of Life Bulb consists of
designing our genetic constructs, assembling plasmids through molecular cloning, testing the expression of
bioluminescence genes, and learning from the results in order to improve future efforts.**

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# INTRODUCTION

Our goal is to produce light in the most ecological way possible. Due to the energy use, greenhouse
gas emissions,
and environmental effects of electrical lighting, we consider biological light production to be the
perfect
candidate as a sustainable alternative. Dinoflagellates were our original inspiration, as they are
responsible for
the famous glowing blue waves that initially attracted our attention. After extensive literature
review, we had a
much better understanding of the current knowledge of bioluminescence, and began planning the design
of our
engineered biological system.

Bioluminescence was our go-to solution as it allows us to generate light in a very efficient manner.
The phenomenon
utilizes an enzyme called **luciferase** along with a substrate called **luciferin** to emit
photons. The
oxidation of luciferin into a high-energy intermediate results in light emission upon decomposition.
This process is
nearly 100% efficient with negligible heat production, resulting in a “cold light”.1
Other methods of producing light not involving combustion nor electricity are:

Comparing Forms of Light Emission

|  | Light by Excitation? | Decays Over Time? | Toxic? | Low Cost? | Easily Improvable? |
| --- | --- | --- | --- | --- | --- |
| Fluorescence | No | Immediate | Maybe | No | No |
| Phosphorescence | No | Yes | Maybe | No | No |
| Non-Biological Chemiluminescence | Yes | Yes | Yes | Maybe | No |
| Bioluminescence | Yes | No | Encapsulated and Recycled | Yes, with Bioreactors | Yes, with Synthetic Biology! |

## BIOLUMINESCENCE

Being able to control bioluminescence has been in the minds of many groups in recent decades. Many
previous studies have expressed luciferases from a variety of species, modified them, and enhanced
them beyond their natural counterparts. The problem is that synthesized luciferin must be externally
added to these organisms to produce light. This is not only costly but also highly inefficient.
That’s why we opted for an autobioluminescent system, meaning that luciferin is produced inside the
organism without the addition of external substrates, allowing it to glow by itself. There are only
two currently elucidated pathways for the complete biosynthesis of luciferin.2, 3

## BACTERIAL BIOLUMINESCENCE

**Properties of the Bacterial Bioluminescent System Luciferase**

* Wavelength: 490 nm
* Optimum pH: 6.8
* Optimum temperature: 23 °C
* Quantum yield: 30%

Bacterial luciferin has been shown to have cytotoxic effects in eukaryotic
organisms.4 Studies encoding both systems in plants show that fungal
bioluminescence produces more light than bacterial bioluminescence.5 Therefore,
we decided to work with this new bioluminescent system recently elucidated in 2018 by
Kotoblay and colleagues.5

## Fungal Bioluminescence

Fungal bioluminescence is the result of a metabolic pathway referred to as the **Caffeic Acid
Cycle**.

![](https://static.igem.wiki/teams/4350/wiki/images/engineering/caffeic-acid-cycle-better.png)

The system starts with the synthesis of hispidin from caffeic acid catalyzed by
hispidin synthase (**HispS**). Then, hispidin is hydroxylated by hispidin-3-hydroxylase
**(H3H)** into the fungal luciferin, 3-hydroxyhispidin. Fungal luciferin is then oxidized by
luciferase (**Luz**), producing a high-energy intermediate that emits photons when degraded into
caffeylpyruvate (fungal oxyluciferin). The cycle starts anew when caffeylpyruvate hydrolase
(**CPH**) recycles oxyluciferin back into caffeic acid, generating pyruvic acid as a byproduct.
Additionally, HispS requires post-translational modification from 4′-phosphopantetheinyl transferase
(**NpgA**) in order to function.

The original study that successfully expressed fungal bioluminescence in a heterologous chassis
utilized genes for the caffeic acid cycle from the fungi *Neonothopanus nambi* and the NpgA
gene from *Aspergillus nidulans*. This approach was expanded upon by Mitiouchkina et al. in
2020, demonstrating successful heterologous expression of bioluminescence in plants.6
Plants naturally produce caffeic acid as part of the lignin and flavonoid synthesis pathways. This
allows for the creation of light-emitting plants without the addition of external substrates through
expression of fungal bioluminescence genes.

# DESIGN

## Chassis Selection

Once we selected a system that is efficient at producing light and doesn't require electrical energy
nor heavy metals, we needed to find other ways of making it “green”. One promising method for the
creation of sustainable biological systems is phototroph engineering. Phototrophs are able to carry
out complex biological processes while fixing carbon dioxide, all while being powered by the sun!
One prominent choice of phototrophic chassis is higher plants, although they possess several
drawbacks that would make a bioluminescent plant lighting system disadvantageous.

Plant drawbacks:

* Although chloroplast engineering is possible, propagation and cell culture are a concern
* Occupy fertile area
* May require constant water supply
* GMO plants are very restricted in many countries

Therefore we examined other options. Phototrophic microorganisms bring with them unique advantages.
Growth in liquid media allows for added versatility by adapting to a container’s shape, and easy
containment of said liquid media allows for improved secondary containment for better biosafety and
a wider range of applications. After deciding upon phototrophic microorganisms as a chassis, we
determined our final goal of having a versatile light source in the form of a growable luminous
liquid that can be incorporated in any environment as a sustainable light source, artistic medium,
or urban design feature.

Microalgae and cyanobacteria were our finalists. Although we considered the advantages of expressing
eukaryotic genes in eukaryotic microalgae, we opted for cyanobacteria for two main reasons. First,
they have a shorter doubling time than common microalgal chassis.7 Second, we aim to
control our bioluminescent system so that it only glows when needed. *Synechocystis* sp. PCC
6803, a species of cyanobacteria, are model organisms for the study of circadian rhythms and the
engineering of systems under circadian control. By demonstrating that this system works in
cyanobacteria, we can continue to try to control the system with confidence through circadian
promoters.

*Synechocystis* sp. PCC 6803 is an ubiquitous chassis in cyanobacterial synthetic biology,
earning the nickname “green *E. coli*”. We decided upon this organism as our phototrophic,
prokayotic chassis, making this the first attempt at expressing the entire fungal bioluminescence
pathway in prokaryotes. Upon success, we can start trying more interesting and challenging chassis
such as saltwater strains.

We obtained a non-motile glucose-tolerant strain from the Hallam Lab in UBC Vancouver. However, we
quickly ran into a problem. *Synechocystis* sp. PCC 6803 doesn’t produce caffeic acid which is
the precursor of fungal luciferin necessary for the production of bioluminescence. We found a
research group that was able to engineer this strain to synthesize caffeic acid.8

We considered this a great finding as our system recycles caffeic acid, and it's possible that it
will start accumulating caffeic acid, feeding the bioluminescence reaction without the need for
external substrates. We managed to contact this group from the University of Copenhagen and they
kindly sent their strain overseas to us!

## Gene Selection

Inspired by the previous attempts of heterologously expressing fungal bioluminescence, we decided to
take the same strategy to increase our chances of success. Sequences for the cDNA of the 4 enzymes
(HispS, H3H, Luz, and CPH) from *N. nambi* and NpgA from *A. nidulans* were obtained from
NCBI.

We explored possible methods of improving the bioluminescence system. Some of our members have
experience with ecology, and suggested we compare literature regarding the relative light
intensities of various bioluminescent fungi. We were informed by mycologists that the fungus that
produces the most visible light was *Panellus stipticus*, which can be found in Canada as well.
All bioluminescent fungi share common bioluminescence genes in a highly-conserved gene cluster, only
differing in certain mutations, duplications, and the presence of p450-coding genes that may
contribute to the process.5 There’s no certainty at the moment regarding the factors
responsible for its increased brightness, but we decided to express the luciferase gene from *P.
stipticus*, as the enzyme’s activity plays a significant role in the intensity of the light
emitted.2

![](https://static.igem.wiki/teams/4350/wiki/images/landing-page/slides/luz-neonm.gif)

3D model of nnLuz from *Neonothopanus nambi* created with alphafold2

![](https://static.igem.wiki/teams/4350/wiki/images/landing-page/slides/luciferase-panellus-stipticus.gif)

3D model of psLuz from *Panellus stipticus* created with alphafold2

On the side, other members dug into the literature to see what other people have accomplished in
improving the system. In the presence of oxygen, fungal luciferase is able to oxidize artificial
luciferin analogs, producing different wavelengths and intensities of light.10 Another
potential method of altering wavelength is through site-directed mutagenesis of the luciferase gene.
One paper explored the effects of site-directed mutagenesis, and determined that most point
mutations resulted in a loss of enzyme function. However, a V49A mutation resulted in a 1.7 times
increase in luminescence intensity, demonstrating the potential for improvement of the
bioluminescent system through mutagenesis. This paper also investigated the structure of luciferase,
speculating the existence of an N-terminal transmembrane domain. The existence of this region was
supported by deletions of up to 37 N-terminal amino acid residues having no effect on luciferase
activity. Inspired by the results of this paper, we decided to express *N. nambi* luciferase
mutants possessing the V49A substitution and 33 N-terminal amino acid deletion in addition to the
wild type *N. nambi* and *P. stipticus* luciferase genes.

Due to how recently the fungal bioluminescence pathway was elucidated, the enzymes are not
well-characterized. For example, fungal luciferase belongs to a novel, undescribed protein family
lacking any known homologs. By expressing different variants of the luciferase gene along with the
rest of the bioluminescence pathway in a novel chassis, we hoped to expand upon the understanding of
heterologous expression of fungal bioluminescence.

## MoClo

After determining the genes to be expressed in *Synechocystis* sp. PCC 6803, we designed our
plasmid constructs. We settled on expression of all fungal bioluminescence genes in single
multi-gene constructs for ease of transformation and expression in *Synechocystis* sp. PCC
6803. Additionally, we utilized a variety of promoters and terminators to minimize the chances of
homologous recombination in our constructs. When considering our plasmid assembly process, we
compared the advantages and disadvantages of traditional BioBrick cloning with Type IIS golden gate
cloning. In comparison to traditional BioBrick cloning, golden gate cloning offers increased
modularity and convenience for large assemblies due to its use of Type IIS enzymes that cut outside
their recognized restriction sites, thus allowing for custom design of fusion sites for large
one-pot, one-step reactions of many inserts simultaneously. The large size of our multi-gene
constructs, the large number of inserts in said constructs, the variety of plasmid designs, and the
future plans for expression with circadian promoters were all factors that contributed to our
decision to use golden gate cloning. This decision was further reinforced by the convenience of the
CyanoGate Kit, a plasmid kit with a large variety of promoters, terminators, and backbones for the
engineering of cyanobacteria through golden gate cloning. CyanoGate specifically utilizes a MoClo
standard, which establishes a specific syntax for the custom fusion sites of assembling MoClo parts
together. Individual basic biological parts in plasmids flanked by Type IIS restriction sites are
classified as level 0 parts, assembly of multiple level 0 parts into a single functioning
transcriptional unit produces a level 1 part, and assembly of multiple level 1 transcriptional units
into a multi-gene construct results in a level 2 part. This assembly process also required vector
backbones for level 0 and level 1 assemblies from the MoClo Toolkit.

The cDNA sequences acquired from NCBI were then codon-optimized using IDT’s integrated software for
expression in *Synechocystis* sp. PCC 6803, and illegal restriction sites for Type IIS
assemblies were removed through silent mutations using Benchling.

![](https://static.igem.wiki/teams/4350/wiki/plasmidmushroomq.gif)

## Circadian Control

The modularity of MoClo allows for the easy integration of circadian promoters into our designed
plasmid constructs. Inducing the expression of bioluminescence through circadian control would allow
for greater manipulation of bioluminescence gene expression to save on resources and possibly
integrate a “charging system” for when light is not available by storing energy and releasing it
later. Although we took these possibilities into consideration in our design process, they were far
outside the scope of our project, and were thus not attempted.

# BUILDING

After determining our genes, assembly method, and plasmid designs, we modified the sequences of our
codon-optimized genes to include 5’ and 3’ flanks for compatibility with golden gate assembly. These
flanks included BbsI restriction sites and fusion sites for assembly of linear fragments into a
level 0 plasmid. Furthermore, they also included BsaI restriction sites and fusion sites for
assembly of level 0 plasmids into level 1 transcriptional units. Following the addition of these
flanks, the genes were synthesized by IDT.

All CyanoGate and MoClo parts were formatted as level 0 plasmids compatible for assembly with our
synthesized fragments. Following the extraction of these parts, we assembled our linear fragments
from IDT in the pAGM9121 universal level 0 acceptor vector from the MoClo Toolkit utilizing the Type
IIS restriction endonucleases BbsI. The assembled level 0 plasmids contained BsaI restriction sites
and the MoClo standard fusion sites for coding sequences that were incorporated into the
IDT-synthesized fragments.

These level 0 plasmids were assembled into level 1 transcriptional units with BsaI, combining
promoters, 5’ UTRs, and terminators from CyanoGate with backbones from the MoClo Toolkit, and our
level 0 gene constructs. The level 1 acceptor backbones utilized produce sticky ends that are
complementary to each other when the level 1 parts are digested with BbsI in a level 2 assembly.
These sticky ends are designed for up to 7 transcriptional units in a level 2 construct, and a dummy
insert is included to produce sticky ends for assembly of our 5 gene constructs. We produced
multi-gene constructs with each variation of luciferase. The transcriptional units were assembled in
backbones in the order of Luz, CPH, H3H, NpgA, HispS. This was chosen based on the conformation of
the genes found in *N. nambi*'s chromosome. We decided to go for a promoter driving the
expression of each gene to help us troubleshoot in the future and also to overexpress as much of the
protein as possible. This system of gene expression was inspired by previous studies with
heterologous expression in cyanobacteria.

Every step of our build process was screened and sequence-verified through antibiotic selection,
blue-white screening, colony PCR, and analytical digestions.

# TESTING

Our first goal is to demonstrate this system is feasible in cyanobacteria. So far there have been no
attempts to express HispS, NpgA, nor CPH in prokaryotes while Luz and H3H have successfully been
expressed with demonstrated enzymatic activity.9, 10

Due to time constraints we decided to not transform cyanobacteria. This process can take weeks due
to their slow growth. Meanwhile, we used our plasmid-carrying *E. coli* to carry out some
tests.

The first test was to see if there was any bioluminescence produced at all. No measurable
bioluminescence was observed in transformed DH5a *E. coli* grown in varying concentrations of
caffeic acid-supplemented media. However, BL21 E. coli are generally used for measuring gene
expression, and DH5a *E. coli* are not as suitable for heterologous gene expression. We also
tried adding varied amounts of caffeic acid and liquid media cultures in a black 96-well plate
donated by Nyoka Design Labs.

No light was observed when looked in a black box (as our plate reader only analyses fluorescence).
We also conducted this experiment with low growth of our level 2 transformed *E. coli*, because
as mentioned above, time constraints didn't allow for further growth. Additionally, the *E.
coli* were incubated at 37 oC, and fungal luciferase is known to inactivate at
temperatures surpassing 30 oC which would greatly explain our results considering we did
not allow the cells to grow enough under 30 oC.

Extraction and visualization of our final construct from transformant colonies through gel
electrophoresis revealed a plasmid construct greater than 10 kilobases (the length could not be
precisely determined as the plasmid construct was larger than all available DNA ladders).

*E. coli* level 2 transformant colonies inoculated in liquid media demonstrated slower growth
than liquid media inoculated with level 1 transformants and wild type *E. coli*, suggesting
that the *E. coli* transformed with all of the bioluminescence genes potentially experienced
metabolic constraints as a result of expressing the bioluminescence genes regulated by strong,
constitutive promoters.

This is a potential indication of bioluminescence gene expression, but further analysis and
expression tests are necessary for verification of expression.

# LEARNING

Our level 1 constructs have no apparent effect on *E. coli* growth while our level 2 and level
T seem to reduce the doubling time of transformed strains. This may indicate that the expression of
these enzymes combined might be intervening with some process in the physiology of *E. coli*.
This may not be the case for cyanobacteria but it's certainly worth consideration in the future.

Future iterations of the engineering design cycle have numerous opportunities to expand upon our
engineered biological system by exploring aspects of engineering bioluminescence outside the scope
of this year’s project. Potential areas to explore include the incorporation of circadian promoters,
the expression of fungal bioluminescence genes spread across multiple smaller plasmid constructs
rather than one large construct, and the transformation of caffeic acid-producing
*Synechocystis* sp. PCC 6803 with fungal bioluminescence genes through natural transformation
or triparental mating.

Further analytical techniques could be utilized for characterizing the expression of fungal
bioluminescent genes in *E. coli* or *Synechocystis* sp. PCC 6803.

We tried conducting an SDS-PAGE analysis but had no results in our WT control and due to time
constraints the experiment was not repeated. Performing this test would allow us to see if the
enzymes are being successfully translated. SDS-PAGE analysis would allow us to compare proteins
present in WT *E. coli*, the level 1 transformants expressing all the 5 single enzymes
individually (as well as the other 3 versions of Luz), and the level 2 transformants. We would
expect a strong band representing each additional enzyme in its corresponding level 1 strain as well
as a combination of all of them in the Level 2 strain.

Protein extraction and fractionation by column chromatography and substrate assays would further
validate protein expression. For measuring the expression of heterologous bioluminescent genes in
*E. coli*, future iterations of the engineering cycle could utilize BL21 *E. coli* rather
than DH5a *E. coli* as we would get much better expression of these exogenous enzymes.

We conducted assays with caffeic acid as well and we noted no bioluminescence. As mentioned before,
this could very well be lack of enough cell density, a good chassis for overexpression of
heterologous genes, and the fact that we never attempted these experiments after leaving the cells
to grow at temperatures under 30oC for sufficient time. In our next iteration of the
engineering cycle, we will design better experiments for *E. coli* and have these in mind for
future attempts with cyanobacteria.

To check for correct expression and troubleshooting we need to conduct metabolite analysis. HPLC and
LC-MS analysis would allow for effective detection of the bioluminescence pathway precursors,
intermediates, and products. Our greatest concern regarding expression of bioluminescence is proper
expression of HispS due to its size, complexity, and post-translational modification. By adding
caffeic acid we would expect the presence of certain intermediates in hispidin synthesis as well as
hispidin in the media. This test can be done with our level 1 strain for HispS as well as our level
2 strains. This method would allow us to see if our enzymes are functional and allow us to try
switching promoters or adding more copies for higher efficiency in the system.

The modularity of golden gate cloning and the successful cloning of all parts allows for easy
modifications to the system by altering promoters, coding sequences, or terminators for expression
of different genes and alteration of gene expression.

Another aspect of the project that could be expanded upon is mutants of the bioluminescence genes.
Site-directed mutagenesis could be utilized to produce different light wavelengths and intensities,
or investigate the activity of enzymes in the bioluminescence pathway.

## REFERENCES

## Open for Reference List

1. (2022) Led. Climate Group. The Climate Group.
2. <https://www.britannica.com/science/bioluminescence>
3. <https://pubs.rsc.org/en/content/articlelanding/2021/CS/D0CS01492C>
4. <https://onlinelibrary.wiley.com/doi/epdf/10.1002/bit.27884?saml_referrer>
5. <https://febs.onlinelibrary.wiley.com/doi/full/10.1016/S0014-5793%2801%2902905-2>
6. <https://www.pnas.org/doi/full/10.1073/pnas.1803615115>
7. <https://nph.onlinelibrary.wiley.com/doi/abs/10.1111/j.1469-8137.1985.tb03634.x>
8. <https://www.sciencedirect.com/science/article/pii/S1096717619303118>
9. <https://pubmed.ncbi.nlm.nih.gov/32917724/>
10. <https://www.pnas.org/doi/full/10.1073/pnas.1803615115>

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{% block header %}Describe the research, experiments, and protocols you used in your iGEM project.{% endblock %}
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## What should this page contain?

---

Describe the research, experiments, and protocols you used in your iGEM project. These should be detailed enough for another team to repeat your experiments.

If you made Parts this year, please remember to put all information, characterization, and measurement data on the Part's Main Page on the [Registry](http://parts.igem.org/Main_Page).

## Inspirations

---

* [2019 Nantes](https://2019.igem.org/Team%3ANantes/Experiments)
* [2019 TU Eindhoven](https://2019.igem.org/Team%3ATU_Eindhoven/Experiments)
* [2019 Mingdao](https://2019.igem.org/Team%3AMingdao/Demonstrate)
* [2020 Amsterdam](https://2020.igem.org/Team%3AAmsterdam/Experiments)
* [2020 NCTU Formosa](https://2020.igem.org/Team%3ANCTU_Formosa/Experiments)
* [2020 USAFA](https://2020.igem.org/Team%3AUSAFA/Experiments)

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[Construction](#construction)
[Validation](#validation)
[Future Plans](#future-plans)
[Interlab](#interlab)
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# CONSTRUCTION

## GOLDEN GATE ASSEMBLY

All Golden Gate Assembly for this project was based on the MoClo Assembly Toolkit from [addgene.org](https://www.addgene.org/). We purchased two kits of parts which allowed us to use the
assembly framework outlined in their paper [here](https://pubmed.ncbi.nlm.nih.gov/21364738/). The
kits are the [MoClo Toolkit](https://www.addgene.org/kits/marillonnet-moclo/)
and the [Cyanogate Kit](https://www.addgene.org/kits/mccormick-cyanogate/). For reference, Level 0
refers to a basic part, Level 1 refers to a transcriptional
unit (Promoter, CDS, and Terminator), and Level 2/T refers to a multi-transcriptional unit expression vector for
*E. coli* / *Synechocystis* sp. PCC 6803
respectively.

For this project, there were a number of specific constructs we aimed to complete. These are listed below, with
plasmid names according to this scheme:
**Level 0:** pBLB\_ (like BULB) + L0- + id (each CDS has its own number)
**Level 1:** pBLB\_ + L1- + id (such as 1a or 3c, number corresponding to CDS and letter to variant of
promoter or terminator)
**Level T:** pBLB\_ + GLOW- + Δx-id (indicating a change from usual scheme of 1a-2a-3a-4a-5a
construct)
Level 2: pBLB\_ + EcoGLOW + Δx-id (indicating a change from usual scheme of 1a-2a-3a-4a-5a construct)

**List of Constructs**

* pBLB\_L0-1
* pBLB\_L0-2
* pBLB\_L0-3
* pBLB\_L0-4
* pBLB\_L0-5
* pBLB\_L0-6
* pBLB\_L0-7
* pBLB\_L0-8
* pBLB\_L1-1a
* pBLB\_L1-1b
* pBLB\_L1-1c
* pBLB\_L1-2a
* pBLB\_L1-3a
* pBLB\_L1-4a
* pBLB\_L1-5a
* pBLB\_L1-6a
* pBLB\_L1-7a
* pBLB\_L1-8a
* pBLB\_GLOW
* pBLB\_GLOWΔ1-1c
* pBLB\_EcoGLOW
* pBLB\_EcoGLOWΔ1-1c

# VALIDATION

## COLONY PCR ANALYSIS - LEVEL 0

**Please note that all agarose electrophoresis gels with a DNA ladder use NEB 1kb Plus, and wells containing
ladder DNA do not count towards numbering.**

![](https://static.igem.wiki/teams/4350/wiki/images/wetlab/firstvalidation.jpg)

**Figure 1.**
**Gels:** Top Left, pBLB\_L0-3; Top Right, pBLB\_L0-1; Bottom Left, pBLB\_L0-4; Bottom Right, pBLB\_L0-2.
**Lanes:** L, NEB 1kb Plus Ladder;
1-4, Colonies 1-4;
5, (+) Control;
6, (-) Control.
[Primer sequences.](/contribution#primers)

In Figure 1, pBLB\_L0-3 and pBLB\_L0-4 (H3H, lane 4 and NpgA, land 3 on left) both show a positive result in
this colony PCR after our first attempt at assembly and transformation.

![](https://static.igem.wiki/teams/4350/wiki/images/wetlab/secondvalidation.jpg)

**Figure 2.**
**Gels:** From top-left to bottom-left; pBLB\_L0-1, pBLB\_L0-2, pBLB\_L0-5, pBLB\_L0-6, pBLB\_L0-7, pBLB\_L0-8.
**Lanes:** L, NEB 1kb Plus Ladder;
1-4, Colonies 1-4;
5, (+) Control;
6, (-) Control.
[Primer sequences.](/contribution#primers)

Figure 2 shows positive results for transformation with pBLB\_L0-1 (gel 1, top; lanes 2-4), pBLB\_L0-6 (gel 2,
bottom; lanes 1-3),
pBLB\_L0-7 (gel 3, top; lanes 1-4), and pBLB\_L0-8 (gel 3, bottom; lanes 2 and 3).

![](https://static.igem.wiki/teams/4350/wiki/images/wetlab/fourthvalidation1.jpg)
![](https://static.igem.wiki/teams/4350/wiki/images/wetlab/fourthvalidation2.jpg)

**Figure 3.**
Plasmid purified from *E. coli* DH5α transformants.
**Lanes:** L, NEB 1kb Plus Ladder;
1, pBLB\_L0-02 Undigested;
2, pBLB\_L0-02 Digested;
3, pBLB\_L0-05 Undigested;
4, pBLB\_L0-05 Digested.
Digestion with NEB BsaI-HFv2.

Figure 3 shows positive results for transformation with pBLB\_L0-2 (lane 2), but not pBLB\_L0-5 (lane 4). We
attained a positive result for pBLB\_L0-2 after our third try.

![](https://static.igem.wiki/teams/4350/wiki/images/wetlab/hisps-valid.jpg)
![](https://static.igem.wiki/teams/4350/wiki/images/wetlab/hisps-valid-sim.jpg)

**Figure 4.**
Plasmid purified from *E. coli* DH5α transformants.
**Lanes:** L, NEB 1kb Plus Ladder;
1, pBLB\_L0-05 Colony 5;
2, pBLB\_L0-05 Colony 9;
3 and 4, irrelevant to this result.
Digestion with NEB BsaI-HFv2.

Finally, a gel (Figure 4) that showcases positive results for pBLB\_L0-5, the final Level 0 piece to be
assembled. Two colonies from a colony PCR conducted on 16 transformant colonies were selected on a whim
based on the fact that their results were slightly different from the rest. Plasmids were extracted and
digested, getting us Figure 4!
In order for us to get this result, we had to undergo at least 9 iterations of the Golden Gate Assembly
protocol. Possible reasons for this could be the oligonucleotides present in the
gBlocks that IDT sent us (thank you!). Furthermore, the gene was split into 2 fragments with a restriction
site to put them together in assembly. Oligonucleotides could have interfered with annealing, as fragments
might have taken the binding overhangs. Nevertheless, our approach
going forward for assembling gBlocks with plasmid backbones will take into account potential impurities, and
involve some sort of purification process or pre-digestion for 2-step assembly.

## DIGESTION ANALYSIS - LEVEL 1

![](https://static.igem.wiki/teams/4350/wiki/images/wetlab/level1-1a6a8a7a.jpg)
![](https://static.igem.wiki/teams/4350/wiki/images/wetlab/level1-1a6a8a7a-sim.jpg)

Plasmid purified from *E. coli* DH5α transformants.
**Lanes:** L, NEB 1kb Plus Ladder;

pBLB\_L1-1a (lane 1), pBLB\_L1-6a (lane 3), pBLB\_L1-7a (lane 5), and pBLB\_L1-8a (lane 7) mini-prepped plasmid DNA
samples were digested, validating presence of
a correct construct in each of these cultures. This result was attained after the first assembly of the
constructs. Would that it were so simple! At least this meant that we were getting better at Golden Gate
Assemblies.

![](https://static.igem.wiki/teams/4350/wiki/images/wetlab/level1-1b1c2a3a4a.jpg)
![](https://static.igem.wiki/teams/4350/wiki/images/wetlab/level1-1b1c2a3a4a-sim.jpg)

**Figure 5.**
Plasmid purified from *E. coli* DH5α transformants.
**Lanes:** L, NEB 1kb Plus Ladder;
1, pBLB\_L1-1b;
2, pBLB\_L1-1c;
3, pBLB\_L1-2a;
4, pBLB\_L1-3a;
5, pBLB\_L1-4a.
Digestion with NEB BbsI-HF.

In Figure 5, pBLB\_L1-1b (lane 1), pBLB\_L1-1c (lane 2), pBLB\_L1-2a (lane 3), pBLB\_L1-3a (lane 4), and pBLB\_L1-4a
(lane 5) mini-prepped plasmid DNA samples were digested, validating presence of
a correct construct in each of these cultures.

## PCR ANALYSIS - LEVEL 2/T

![](https://static.igem.wiki/teams/4350/wiki/images/wetlab/colony-pcr-level2.jpg)

**Figure 6.**
**Lanes:** L, NEB 1kb Plus Ladder;
1 Negative Colony PCR;
2, Colony PCR of *E. coli* transformed with pBLB\_EcoGLOWΔ1-1c w/ pCAT.000 Fwd and Rev primers;
3, Colony PCR of *E. coli* transformed with pBLB\_EcoGLOWΔ1-1c 2/ pCAT.000 Fwd and PlacIQ Rev
primers.
[Primer sequences.](/contribution#primers)

This (Figure 6) is where the lack of time kicks in! We were without lab for this section, so we had to borrow a
classroom lab on a week with no lab classes just to get this one result. We ran a colony PCR on a colony of
*E. coli* which supposedly contained our final Level 2 construct,
pBLB\_EcoGLOWΔ1-1c. This result shows a possible successful transformation of our pBLB\_EcoGLOWΔ1-1c
plasmid, which is 18kb!

The Wet Lab's next step here would be to perform an SDS-PAGE with a wild-type *E. coli* in order to see if
any new proteins are being expressed. We would then move on to attempting to fractionate protein extracts
of wild-type and transformed *E. coli* and running them on SDS-PAGE gels to see if we could see a more
obvious band where we expect to see our target enzymes.

## CAFFEIC ACID TRIALS

![](https://static.igem.wiki/teams/4350/wiki/images/pfps/caffeictrialsgrowth.jpg)

Growth comparison of *E. coli* transformants. From left to right: pBLB\_EcoGLOW, pBLB\_EcoGLOW, pBLB\_GLOW,
pBLB\_GLOW, Wild Type *E. coli* DH5α.

Our original plan was to clone pBLB\_GLOW in *E. coli* first, to get a high yield of purified plasmid DNA
for transformation into *Synechocystis* sp. PCC 6803. However, due to time constraints, we decided to use
these transformants to test gene expression of the plasmid first.

As *E. coli* do not produce caffeic acid, we had to provide it in order to be able to detect activity of
our design. If all enzymes work (excluding CPH) we would expect to see the emission of light from the cells.

To test this theory, we designed an assay with
varying concentrations of caffeic acid to be provided to living *E. coli*. These *E. coli*
transformants expressing pBLB\_GLOW were combined with caffeic acid in a 96-well plate and placed into a
pitch-black box with a hole for a camera to detect light emission.
To the same effect, we designated additional wells to the addition of *E. coli* expressing individual
transcriptional units (Level 1 assemblies) in case the metabolic load of all 5 enzymes was too much for our
strain of *E. coli* DH5α.

![](https://static.igem.wiki/teams/4350/wiki/images/pfps/failure.jpg)

Shown by the image above, no luminescence was detected.

# FUTURE PLANS

## Expression Quantification

An absolutely major limitation for getting results for the duration of this project was always time. As a
first-year team, we spent a lot of time
getting a lab, getting basic materials, ordering *E. coli* and parts from [addgene.org](https://www.addgene.org/). This took a tremendous amount of time away from working on
constructs and testing them thoroughly.
Furthermore, near the end of the season and as school started again, we lost the ability to work in the lab.
This was because the lab space we were given was turned into
a classroom for the semester. This left us with a couple weeks where we couldn't do much work, and any work we
did do was in a different lab!

Despite this hardship, we believe that this project still has potential. This season, we were not able to make
any organism glow using fungal bioluminescent genes,
but we can still see the path forward!

First, we would like to undertake more rigorous validation of the Level 2 and Level T constructs, pBLB\_EcoGLOW
and pBLB\_Glow, respectively. This would involve:

1. Expression of pBLB\_EcoGLOW and variants in a strain of *E. coli* like BL21 to express high amounts of
   protein
2. Protein Extraction and Fractionation by Column Chromatography
3. SDS-PAGE Analysis of Fractions
4. Further Fractionation and Purification
5. Substrate Assays
6. Individual Expression and Purification of Enzymes

1. Expressing our constructs in a strain like *E. coli* BL21 instead of DH5alpha would facilitate the
extraction of our target proteins. This would allow
us to better quantify the expression of our protein and leave us with some flexibility with yield.

2. We were not able to procure any materials dedicated to extract proteins for analysis. For this reason, we
would like to dedicate budget to purchase them.
We would like to extract our protein from our cultures so that we can fractionate them using a method such as
His-tagging. This way, we would be able to be more
precise with quantifying expression of our genes of interest.

3. Next, we would use an SDS-PAGE to visualize the presence of our target protein in comparison to a wild-type
negative control. If a band appears in our transformed
which does not appear in the wild-type, it is likely that it is our target protein. We can then fractionate the
fraction it was located in to get a pure sample.

4. After identifying which fraction our protein belongs to, we could either use a different column
chromatography method or move to HP-LC analysis to purify our
target protein.

5. After purification, we would be able to use our pure protein samples to guide an assay. This assay would be
dependant on our primary substrate: caffeic acid.
After introducing pathway proteins to the substrate, we can analyse the conversion of substrate into product
using LCMS-MS analysis on the resulting solution.

6. To assist the characterization of the activity of enzymes, we can express each enzyme separately using our
pBLB\_L1 transcriptional unit variants in *E. coli* BL21 and purify them to get each enzyme on their own.
Then, we can do assays such as the following:

* Conversion of Caffeic Acid into Hispidin by HispS enzyme with presence or absence of the NpgA enzyme.
* Conversion of Hispidin into 3-Hydroxyhispidin by H3H enzyme, using substrate purified from above reaction
  products.
* Luminescence of the nnLuz and variants or psLuz enzymes by catalyzing conversion of 3-Hydroxyhispidin into
  Fungal Oxyluciferin
* Cyclic conversion of Caffeic Acid → Hispidin → 3-Hydroxyhispidin →
  Oxyluciferin → Caffeic Acid using all enzymes in the cycle.
* With pure caffeic acid as a substrate, characterize the whole bioluminescent cycle by introducing one enzyme
  at a time.
* With proper time, quantification of quantum yield and overall light emission using specialized instruments
  and software.
* Comparison of nnLuz, nnLuz variants, and psLuz's quantum yields *in vitro* and *in vivo* (*E.
  coli*)

Then, with credence to the whole project's purpose, or even at the same time with a big enough lab, we will
pioneer the expression of the fungal bioluminescent biosynthesis pathway
in *Synechocystis* sp. PCC 6803, as well as take measurements of quantum yield and overall light emission
*in vivo*. Below is a picture of some fun we had with the calibration plate,
post-calibration.

For the Caffeic Acid Trials, we would like to attempt this again using *E. coli* BL21 transformants of
pBLB\_GLOW or pBLB\_EcoGLOW, as well as *Synechocystis* sp. PCC 6803 transformants of pBLB\_GLOW. We strongly
believe that it will shed some light!

# INTERLAB CONTRIBUTION

## CALIBRATION

Our team this year decided to attempt every protocol of this year's Interlab Study. However, due to extremely
transformation efficiency
for unknown reasons and a severe lack of time, we were not able to complete any protocol other than the
calibration measurements.

![](https://static.igem.wiki/teams/4350/wiki/images/wetlab/calibration.jpg)

The black 96-well plate used to submit calibration data for the 2022 iGEM Interlab Study.

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