iGEM 2021 — Team Northern_BC

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# Team:Northern BC/Notebook

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[Home](https://2021.igem.org/Team%3ANorthern_BC)

Project

[Description](https://2021.igem.org/Team%3ANorthern_BC/Description)
[Collaborations](https://2021.igem.org/Team%3ANorthern_BC/Collaborations)
[Contributions](https://2021.igem.org/Team%3ANorthern_BC/Contribution)
[Model](https://2021.igem.org/Team%3ANorthern_BC/Model)
[Proposed Implementation](https://2021.igem.org/Team%3ANorthern_BC/Implementation)

Human Practices

[Human Practices](https://2021.igem.org/Team%3ANorthern_BC/Human_Practices)
[Communication](https://2021.igem.org/Team%3ANorthern_BC/Communication)

Wet Lab

[Notebook](https://2021.igem.org/Team%3ANorthern_BC/Notebook)
[Experiments](https://2021.igem.org/Team%3ANorthern_BC/Experiments)
[Engineering](https://2021.igem.org/Team%3ANorthern_BC/Engineering)
[Parts](https://2021.igem.org/Team%3ANorthern_BC/Parts)
[Results](https://2021.igem.org/Team%3ANorthern_BC/Results)
[Safety](https://2021.igem.org/Team%3ANorthern_BC/Safety)

Team

[Members](https://2021.igem.org/Team%3ANorthern_BC/Team)
[Attributes](https://2021.igem.org/Team%3ANorthern_BC/Attributions)

# Notebook

#### Click on dates to view activities.

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# Solve real world problems with synthetic biology

Contact us

[Home](https://2021.igem.org/Team%3ANorthern_BC)
 [Project](https://2021.igem.org/Team%3ANorthern_BC/Description)
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 [Wet Lab](https://2021.igem.org/Team%3ANorthern_BC/Notebook)
 [Team](https://2021.igem.org/Team%3ANorthern_BC/Team)

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# Team:Northern BC/Engineering

![](https://static.igem.org/mediawiki/2021/1/17/T--Northern_BC--teamLogo.png)

[Home](https://2021.igem.org/Team%3ANorthern_BC)

Project

[Description](https://2021.igem.org/Team%3ANorthern_BC/Description)
[Collaborations](https://2021.igem.org/Team%3ANorthern_BC/Collaborations)
[Contributions](https://2021.igem.org/Team%3ANorthern_BC/Contribution)
[Model](https://2021.igem.org/Team%3ANorthern_BC/Model)
[Proposed Implementation](https://2021.igem.org/Team%3ANorthern_BC/Implementation)

Human Practices

[Human Practices](https://2021.igem.org/Team%3ANorthern_BC/Human_Practices)
[Communication](https://2021.igem.org/Team%3ANorthern_BC/Communication)

Wet Lab

[Notebook](https://2021.igem.org/Team%3ANorthern_BC/Notebook)
[Experiments](https://2021.igem.org/Team%3ANorthern_BC/Experiments)
[Engineering](https://2021.igem.org/Team%3ANorthern_BC/Engineering)
[Parts](https://2021.igem.org/Team%3ANorthern_BC/Parts)
[Results](https://2021.igem.org/Team%3ANorthern_BC/Results)
[Safety](https://2021.igem.org/Team%3ANorthern_BC/Safety)

Team

[Members](https://2021.igem.org/Team%3ANorthern_BC/Team)
[Attributes](https://2021.igem.org/Team%3ANorthern_BC/Attributions)

# The engineering and design cycle was of instrumental importance to the successful creation of the D-tector and the composite part [BBa\_K3957000](http://parts.igem.org/Part%3ABBa_K3957000).

### Our target for the project is to create a cheaper and less machinery intensive vitamin D sensor that could accurately measure the concentration of vitamin D in a blood serum sample such that the deficiency can be classified.

### Preliminary research was done into the current methodologies to tackle vitamin D detection of measurement. It was determined that, especially for northern British Columbia, the major limiting factor was an unavailability of scientific equipment to perform such tests; therefore our system must make use of minimal and accessible scientific equipment. After some research, two possible ideas to address the problem were formulated: an expression system that takes advantage of natural metabolic pathways, and a chimeric GPCR.

# The Engineering Cycle for GPCR

### GPCR Cycle

* #### Research
* #### Imagine
* #### Design

# The Human Metabolic pathway

### Cycle 1

* #### Preface
* #### Research
* #### Imagine
* #### Design
* #### Improve

# Megalin, Cubilin and the purify step

### Cycle 2

* #### Preface
* #### Research
* #### Imagine
* #### Design
* #### Improve

# 25-hydroxyvitamin D-1 alpha hydroxylase and the Activate step

### Cycle 3

* #### Preface
* #### Research
* #### Imagine
* #### Design
* #### Improve
* #### Learn
* #### Testing

# VDR-FRET and the Detect step

### Cycle 4

* #### Preface
* #### Research
* #### Imagine
* #### Design
* #### Improve
* #### Learn
* #### Testing

# Plasmid design

### Cycle 5

* #### Preface
* #### Research
* #### Imagine
* #### Design
* #### Improve

# Protein production management

### Cycle 6

* #### Preface
* #### Research
* #### Imagine
* #### Design
* #### Improve
* #### Testing

# At home vs benchtop

### Cycle 7

* #### Preface
* #### Research
* #### Imagine
* #### Design

#### 1. Wacker, M; Holick, M. F. Vitamin D-Effects on Skeletal and Extraskeletal Health and the Need for Supplementation. Nutr. 2013, 14(5), 111-148. 10.3390/nu5010111

#### 2. Cheskis, B.; Freedman, L. P. Ligand modulates the conversion of DNA-bound vitamin D3 receptor (VDR) homodimers into VDR-retinoid X receptor heterodimers. Mol. Cell. Biol.. 1994, 14(5), 3329-3338. https://doi.org/10.1128/MCB.14.5.3329

#### 3. Protein. <https://www.ncbi.nlm.nih.gov/protein>(Accessed 27-09-2021).

#### 4. Vemulapati, S.; Rey, E.; O’Dell, D.; Mehta, S.; Erickson, D. A quantitative point-of-need assay for the assessment of vitamin D3 deficiency. Nat. 2017, 7, 14142-14150. <https://doi.org/10.1038/s41598-017-13044-5>

#### 5. Sawada, N.; Sakaki, T.; Kitanaka, S.; Takeyama, K.; Kato, S.; Inouye, K. Enzymatic properties of human 25-hydroxyvitamin D3 1alpha-hydroxylase coexpression with adrenodoxin and NADPH-adrenodoxin reductase in escherichia coli. Eur. J. Biochem. 1999, 265(3), 950. doi: 10.1046/j.1432-1327.1999.00794.x

#### 6. Imperial 2007. T7 promoter. <http://parts.igem.org/wiki/index.php?title=Part:BBa_I719005> . (Accessed 27-09-2021).

#### 7. Anderson, J. Ribosome Binding Site Family Member. Part:BBa J61100 - parts.igem.org. (Accessed 27-09-2021).

#### 8. Shetty, R. Double terminator. [http://parts.igem.org/Part:BBa\_B0015](http://parts.igem.org/Part%3ABBa_B0015). (Accessed 27-09-2021).

#### 9. Klupt, K. Vitamin D receptor binding protein with cysteine modification(s) to bind to a biosensor and FRET to. [http://parts.igem.org/Part:BBa\_K3515011](http://parts.igem.org/Part%3ABBa_K3515011). (Accessed 27-09-2021).

![close Button](https://static.igem.org/mediawiki/2021/a/ae/T--Northern_BC--closeIcon.svg)

We looked into various GPCRs in yeast and found that the yeast
pheromone receptor STE2 was most suitable, as it had previously
been targeted for directed evolution in the literature.

![close Button](https://static.igem.org/mediawiki/2021/a/ae/T--Northern_BC--closeIcon.svg)

We need to develop a mutant with preferential binding to DBP.

- The natural ligand of STE2 is alpha-factor (a small
peptide); as such, it is more feasible to target DBP (also a
peptide) than vitamin D directly.

![close Button](https://static.igem.org/mediawiki/2021/a/ae/T--Northern_BC--closeIcon.svg)

1) A Monte-Carlo system would be developed to optimize the binding
affinity of DBP to the yeast pheromone receptor.

2) This system would include a loop of the following activities:

-A program to randomly mutate amino acid regions of the STE2 GPCR
that are key to binding activity
-A program/webservice to model the mutated STE2 GPCR receptor
region, designating three dimensional atomic coordinates
-A program/webservice to check the binding affinity of DBP in
direct comparison to the binding affinity of alpha-factorA program
to receive scoring values for vitamin D / alpha-factor binding and
determine which version of the GPCR will move forward to the next
iteration.

3) We tried using computer-aided directed evolution of enzymes (CADEE). We attempted to use the [CADEE program](https://github.com/kamerlinlab/cadee) developed by the Kamerlin lab from Uppsala University; unfortunately, we were unable to get the program to function.

4) At this point, we decided to shift focus to our other idea: a biosensor that mimicked vitamin D metabolism.

![close Button](https://static.igem.org/mediawiki/2021/a/ae/T--Northern_BC--closeIcon.svg)

Biomimicry was a natural place to start our research, and thus we
began by studying the metabolic processes involving vitamin D.

![close Button](https://static.igem.org/mediawiki/2021/a/ae/T--Northern_BC--closeIcon.svg)

We started by researching and understanding the metabolic pathway
vitamin D follows in the human body. There are into four major
steps1, 2:

- 25-hydroxyvitamin D (25(OH)D) is bound to a storage protein in
the bloodstream
- 25(OH)D enters a kidney cell through large multidomain proteins
called megalin and cubilin, which separates the vitamin from its
storage protein
- 25-hydroxyvitamin D-1 alpha hydroxylase
(CYP27B1) converts 25(OH)D to 1,25-dihydroxyvitamin D
(1,25(OH)2D)
- 1,25(OH)2D binds to vitamin D receptor’s (VDR) ligand
binding domain, which then induces downstream effects by
dimerizing with retinoid X receptor (RXR) and then binding to DNA
enhancer sequences.

![close Button](https://static.igem.org/mediawiki/2021/a/ae/T--Northern_BC--closeIcon.svg)

1) We could express all the proteins necessary for vitamin D gene
regulation in the human body in *E. coli* with the endpoint enhancer
sequences promoting a colorimetric or fluorescent protein. Change
in colour or fluorescence could be the determinator of vitamin D
concentration.
2) We initially wanted it to be an at-home test, and so we considered a colour change or agglutination. Fluorescence requires lab
equipment (which precipitated our shift to a benchtop system)

![close Button](https://static.igem.org/mediawiki/2021/a/ae/T--Northern_BC--closeIcon.svg)

1) The following proteins (and associated DNA base pair length)
would have to be expressed in 1-2 plasmids due to total length3

- Megalin (13 965 bp)
- Cubilin (10 869 bp)
- 25-hydroxyvitamin D-1 alpha hydroxylase (1 524 bp)
- VDR (1 281 bp)
- RXR (1 386 bp)
- Fluorescent protein (mCherry: 708 bp)
- Each protein would individually need to be tested for
functionality
- BioBrick, Golden Gate, and/or Gibson assembly could be
used.

![close Button](https://static.igem.org/mediawiki/2021/a/ae/T--Northern_BC--closeIcon.svg)

To follow this pathway we would need to express, ligate and
transform a multitude of proteins; this system was inelegant and
unfeasible, especially given our resources and timeline.
Therefore, a better system was needed to be developed to simplify
the signal transduction process.

![close Button](https://static.igem.org/mediawiki/2021/a/ae/T--Northern_BC--closeIcon.svg)

1) In the preceding plan, megalin and cubilin are the two largest
proteins. The combination of these two would use more than the 20
000 free nucleotides IDT gives each team. Additionally, it was
unknown how these large cell membrane proteins would interact
within the gram negative bacterial outer/inner membrane system.

2) A new way to separate vitamin D from its storage protein was
needed.

![close Button](https://static.igem.org/mediawiki/2021/a/ae/T--Northern_BC--closeIcon.svg)

1) We were unable to find alternative protein complexes that could
separate the vitamin D.
2) Research into the current vitamin D testing methods revealed
that the separation of vitamin D from blood serum was not the cost
and equipment limiting step.
Organic solvents, such as acetonitrile, are currently used to
separate vitamin D from its binding protein.

![close Button](https://static.igem.org/mediawiki/2021/a/ae/T--Northern_BC--closeIcon.svg)

Vitamin D could be purified from a blood serum sample and applied
to our cells, which produce a signal.

![close Button](https://static.igem.org/mediawiki/2021/a/ae/T--Northern_BC--closeIcon.svg)

Using organic solvents, we no longer have to express megalin and
cubilin and the only change to the project design is a new purify
step in the protocol.

![close Button](https://static.igem.org/mediawiki/2021/a/ae/T--Northern_BC--closeIcon.svg)

No further improvements have been made and this step is now deemed
the “purify” step in vitamin D detection.

![close Button](https://static.igem.org/mediawiki/2021/a/ae/T--Northern_BC--closeIcon.svg)

In the human body VDR interacts with 1,25(OH)2D;
therefore, for our project to sense the storage form of vitamin D
(25(OH)D), it must be hydroxylated.

![close Button](https://static.igem.org/mediawiki/2021/a/ae/T--Northern_BC--closeIcon.svg)

Sawada, N et al. (5) showed that 25-hydroxyvitamin D-1 alpha
hydroxylase could be expressed in *E. coli* without additive
proteins or molecules.

![close Button](https://static.igem.org/mediawiki/2021/a/ae/T--Northern_BC--closeIcon.svg)

We could express 25-hydroxyvitamin D-1 alpha hydroxylase in *E.
coli* so that the storage form of vitamin D can be converted such that it can interact with VDR.

![close Button](https://static.igem.org/mediawiki/2021/a/ae/T--Northern_BC--closeIcon.svg)

1) The genetic code of 25-hydroxyvitamin D-1 alpha hydroxylase was
taken from [NCBI](https://www.ncbi.nlm.nih.gov/gene/1594) by converting the protein amino
acid sequence to nucleotides; this way, introns would not need to
be removed4.
&nbsp- The genetic code was codon optimized to 14% in SnapGene based on *E.
coli* codon tables.

2) A His-tag was added for protein purification.
3) Based on available restriction enzymes, BioBrick was chosen as
a gene insertion method
- Multiple restriction enzyme sites had to be removed so that
this gene would be BioBrick compatible. This was done by manually changing the nucleotides
while maintaining the codons adjacent to the restriction enzyme
site.

4) Since we no longer needed the IDT free nucleotides for megalin
and cubilin, we were able to make “functional” units of
25-hydroxyvitamin D-1 alpha hydroxylase. This means that in one
BioBrick part we had:
- BioBrick prefixes and suffixes
- A T7 promoter site ([BBa\_I719005](http://parts.igem.org/Part%3ABBa_I719005))6. The promoter was chosen based on the engineering
cycle for protein production management.
- A ribosome binding site ([BBa\_J61100](http://parts.igem.org/Part%3ABBa_J61100))7
. The RBS was chosen based on maximal efficiency.

- The gene
- A His-tag
- A terminator ([BBa\_B0015](http://parts.igem.org/Part%3ABBa_B0015))8
. A double terminator was chosen it was well characterized on the iGEM parts page

![close Button](https://static.igem.org/mediawiki/2021/a/ae/T--Northern_BC--closeIcon.svg)

1) Once the BioBrick was completed, the part was sent to IDT and
it was determined the complexity score was too high. To create our
project, we split the part down the middle and ordered two
separate parts with lower complexity scores individually.
- We then had two separate parts that needed to be combined; we
determined that PCR SOEing would be an effective technique to
accomplish this.

![close Button](https://static.igem.org/mediawiki/2021/a/ae/T--Northern_BC--closeIcon.svg)

1) After testing, an effective PCR SOEing protocol was determined.
Unfortunately, the product was unable to be amplified through
regular PCR.
- After careful consideration of the goals of the project and a
workable timeline it was determined that there was no need for the
creation of a successful PCR SOEing product amplification
protocol, and that the original PCR SOEing product would be enough
for future BioBrick ligations.

![close Button](https://static.igem.org/mediawiki/2021/a/ae/T--Northern_BC--closeIcon.svg)

1) To confirm the ligation and transformation of the
25-hydroxyvitamin D-1 alpha hydroxylase BioBrick functional unit,
a colony PCR was performed
- Colony PCR suggested correct ligation of 25-hydroxyvitamin D-1
alpha hydroxylase BioBrick functional unit in pSB1C3

2) Sequencing was done to confirm DNA sequence
- There was a deletion/frameshift upstream from the His-tag
invalidating the part
- The 25-hydroxyvitamin D-1 alpha hydroxylase BioBrick functional
unit was set aside so that we could focus on testing and
characterizing [BBa\_K3957000](http://parts.igem.org/Part%3ABBa_K3957000).

![close Button](https://static.igem.org/mediawiki/2021/a/ae/T--Northern_BC--closeIcon.svg)

To detect the concentration of vitamin D in a sample following the
natural mechanism, three proteins would need to be expressed and
tested: VDR, RXR, and an expressible fluorescent protein. To
transform, express and test all these proteins would be an
unreasonable undertaking for our team so a better solution would
need to be found.

![close Button](https://static.igem.org/mediawiki/2021/a/ae/T--Northern_BC--closeIcon.svg)

1) After we searched the iGEM Parts Registry, we found [a chimeric
protein consisting of the ligand binding domain from VDR, mCherry,
and mNeon green](http://parts.igem.org/Part%3ABBa_K3515011).
- This chimeric protein worked under the foundations of Förster
resonance energy transfer or FRET for short.
- The chimeric protein was made in the program [Chimera](https://www.cgl.ucsf.edu/chimera/) and had not
been expressed before, but after careful consideration we decided
it was the best move forward for the project.
- Multiple communications were made with the creators
of the part from the [2020 Queen's iGEM team](https://2020.igem.org/Team%3AQueens_Canada) to get a deeper understanding of its inception and
intended use.

2) Inspection of the measurement abilities of FRET systems was
evaluated, and it was determined that the FRET protocol could be
able to measure the levels of vitamin D in a sample.

![close Button](https://static.igem.org/mediawiki/2021/a/ae/T--Northern_BC--closeIcon.svg)

After the 25(OH)D is converted to 1,25(OH)2D in the
activate step it can immediately interact with the VDR-FRET to
produce a signal thus creating the “detect” step in our project.

![close Button](https://static.igem.org/mediawiki/2021/a/ae/T--Northern_BC--closeIcon.svg)

1) The genetic code of BBa\_K3515011 was copied from its [iGEM Part
page](http://parts.igem.org/Part%3ABBa_K3515011).9
- The genetic code was codon optimized to 14% based on *E. coli*
codon tables.
- On the parts sequence there were three stop codons in the
reading frame that were removed. These stop codons were after each
domain used to create the chimeric protein: mNeon green, the
ligand binding domain of VDR, and mCherry.

2) A His-tag was added for protein purification.

3) Based on available restriction enzymes, BioBrick was chosen as
a gene insertion method
- Multiple restriction enzyme sites had to be removed so that this
gene would be BioBrick compatible. This was done by hand changing the nucleotides while
maintaining the codons adjacent to the restriction enzyme site.

4) Since we no longer needed the IDT free nucleotides for megalin
and cubilin, we were able to make “functional” units of
our chimeric part. This means that in one
BioBrick part we had:
- BioBrick prefixes and suffixes
- A T7 promoter site ([BBa\_I719005](http://parts.igem.org/Part%3ABBa_I719005))6. The promoter was chosen based on the engineering
cycle for protein production management.
- A ribosome binding site ([BBa\_J61100](http://parts.igem.org/Part%3ABBa_J61100))7
. The RBS was chosen based on maximal efficiency.

- The gene
- A His-tag
- A terminator ([BBa\_B0015](http://parts.igem.org/Part%3ABBa_B0015))8
. A double terminator was chosen it was well characterized on the iGEM parts page

![close Button](https://static.igem.org/mediawiki/2021/a/ae/T--Northern_BC--closeIcon.svg)

1) Once the modifications to the sequence were completed the part was sent to IDT, and
it was determined the complexity score was too high. So we split
the part down the middle and ordered two separate parts with lower
complexity scores individually.
- Now that we had 2 separate parts that needed to be combined, we
determined that PCR SOEing would be an effective technique to use.

2) After testing an effective PCR SOEing protocol was determined.
Unfortunately, the PCR SOEing product was unable to be amplified
through regular PCR.
- After careful consideration of the project goals and a workable
timeline, it was determined that there was no need for the
creation of a successful PCR SOEing product amplification
protocol, and that the original PCR SOEing product would be enough
for future biobrick ligations.

![close Button](https://static.igem.org/mediawiki/2021/a/ae/T--Northern_BC--closeIcon.svg)

1) After some testing it was revealed that the fluorescence reader
we were using, the SynergyTM 2 Multi-Mode Microplate Reader
, was unable to test the fluorescence
of mNeonGreen with the filters available to us. It can detect the fluorescence of mCherry, but it
was unable to measure at the maximal wavelength. As for the FRET
system, the plate reader can accurately excite mNeonGreen and measure
the emission of mCherry.
- This means we could only use the data from FRET to describe our
protein rather than each individual fluorophore.

2) It was revealed that VDR-FRET was able to provide a detectable
background fluorescence; however, the FRET signal did not change
with increasing and decreasing 1,25-dihydroxyvitamin D
concentrations, suggesting that the VDR-LBD is not able to
interact with the 1,25-dihydroxyvitamin D to cause conformational
change.

![close Button](https://static.igem.org/mediawiki/2021/a/ae/T--Northern_BC--closeIcon.svg)

1) To confirm the ligation and transformation of the VDR-FRET
biobrick functional unit, colony PCR was performed.
- Colony PCR suggested correct ligation of the 25-hydroxyvitamin
D-1 alpha hydroxylase BioBrick functional unit

2) Sequencing was done to confirm the DNA sequence.
- There was a codon change in mNeonGreen at the 72nd codon: His → Asn

![close Button](https://static.igem.org/mediawiki/2021/a/ae/T--Northern_BC--closeIcon.svg)

With limited resources available, the decision on which plasmid to
use was based on what was available to our lab.

![close Button](https://static.igem.org/mediawiki/2021/a/ae/T--Northern_BC--closeIcon.svg)

1) Looking at our inventory, the 2070 bp plasmid [pSB1C3](http://parts.igem.org/Part%3ApSB1C3) was
available and included a BioBrick prefix/suffix zone
- We had a pSB1C3 stock with a red fluorescent
protein (RFP) from a previous year's distribution kit.
-- The RFP was inserted in the BioBrick site, so we planned to use
restriction enzymes and gel purification a linearized pSB1C3 with
the correct cut BioBrick sites for the addition of our BioBrick
functional.

2) We also had a stock of chloramphenicol from previous iGEM years
which is the same antibiotic resistance given by pSB1C3.

![close Button](https://static.igem.org/mediawiki/2021/a/ae/T--Northern_BC--closeIcon.svg)

We could create two plasmid stocks, one with the VDR-FRET BioBrick
inserted into pSB1C3, and another with both VDR-FRET and the
25-hydroxyvitamin D-1 alpha hydroxylase. This allowed us to test
the effects of 25(OH)D and 1,25(OH)2D on the whole
system and the individual functionality of VDR-FRET.

![close Button](https://static.igem.org/mediawiki/2021/a/ae/T--Northern_BC--closeIcon.svg)

1) We removed the RFP from pSB1C3 by digesting the plasmid with
SpeI and EcoRI.
2) VDR-FRET was prepared for ligation into the plasmid by
digesting with EcoRI and SpeI
3) 25-hydroxyvitamin D-1 alpha hydroxylase was prepared for
ligation into the plasmid by digesting with EcoRI and SpeI.

![close Button](https://static.igem.org/mediawiki/2021/a/ae/T--Northern_BC--closeIcon.svg)

We tried a 10 minute ligation combining pSB1C3 and VDR-FRET, but
it was unsuccessful.
- It was determined that since pSB1C3 is only 2070 bp and
VDR-FRET 2426 bp, the ligation will be less efficient than
expected, so we switched to an overnight ligation. The overnight ligation provided satisfactory results that
allowed us to progress with the project.

![close Button](https://static.igem.org/mediawiki/2021/a/ae/T--Northern_BC--closeIcon.svg)

This project revolves around the production of large quantities of
proteins from which we can purify and use to test samples. A
strong promoter made sense to use in our cell cultures.

![close Button](https://static.igem.org/mediawiki/2021/a/ae/T--Northern_BC--closeIcon.svg)

1) Research for this engineering goal came in two parts: what
could be done, and what can be done.
2) Previous iGEM teams had a supply of competent DH5α cells
3) Through generous donation by the Murray and Rader laboratories
at UNBC, the iGEM team was able to obtain both DH5α and BL21(DE3)
non-competent *E. coli* cells.
4) Based on this, the viral T7 promoter was chosen to enhance the
production of our protein. This is due to the fact DH5α cells lack
the ability to produce T7 polymerase; whereas, BL21(DE3) cells
have the T7 polymerase under inducible control.

![close Button](https://static.igem.org/mediawiki/2021/a/ae/T--Northern_BC--closeIcon.svg)

With two cell lines and differential abilities to express the
proteins, the DH5α cells could be used to create a storage cell
line and the BL21(DE3) cells could act as "factories" to produce our proteins.

![close Button](https://static.igem.org/mediawiki/2021/a/ae/T--Northern_BC--closeIcon.svg)

1) It was determined that the cloning and transforming effort
would be done in the DH5α cells since they cannot produce the
desired proteins.
2) The induction of protein production would be done in the
BL21(DE3) cell line.

![close Button](https://static.igem.org/mediawiki/2021/a/ae/T--Northern_BC--closeIcon.svg)

Transformation of the DH5α cells with the previous iGEM team's
competent DH5α cells did not produce results.
- We tested the antibiotic resistance on the plates, the plasmid
itself, and the protocol used, and it was determined that on the
cell's creation their competency was too low to transform with
reasonable amounts of plasmid. To push through this, the donated DH5α and BL21(DE3)
non-competent cells were used to create fresh competent cells
which had a high enough competency to transform with our plasmid.

![close Button](https://static.igem.org/mediawiki/2021/a/ae/T--Northern_BC--closeIcon.svg)

During protein production a multitude of cell lines were grown
up in an LB broth: an IPTG induced transformed BL21(DE3) cell
line, an uninduced transformed BL21(DE3) cell line, and an
uninduced untransformed BL21(DE3) cell line. After growing up the cell lines, both the uninduced and induced
transformed BL21(DE3) cells had protein production. Further research suggested that the LB broth we used for the
culture growth medium contained lactose, which was
enough to induce the cultures.

![close Button](https://static.igem.org/mediawiki/2021/a/ae/T--Northern_BC--closeIcon.svg)

Originally, an at-home test would be the gold standard for project
design; However, one of the main reasons behind the cost of
vitamin D testing in northern British Columbia is the complications
associated with getting one. An at-home test could side step these; however, the concept of a benchtop kit for laboratory
use was not completely taken off the table.

![close Button](https://static.igem.org/mediawiki/2021/a/ae/T--Northern_BC--closeIcon.svg)

Originally, an at-home test kit was able to fit into our project
when megalin, cubilin, RXR and VDR were still on the table;
unfortunately, after the decision to move to VDR-FRET and the
necessity for a fluorescence reader, the idea of an at- home kit
was no longer feasible.

![close Button](https://static.igem.org/mediawiki/2021/a/ae/T--Northern_BC--closeIcon.svg)

The project could be a premade kit sold to a local health
facility. Purified protein samples could be stored and frozen in a
well plate, such that a set volume of blood serum sample would be
added to a well and the fluorescence could be measured.

![close Button](https://static.igem.org/mediawiki/2021/a/ae/T--Northern_BC--closeIcon.svg)

The project never made it to a proof of concept as VDR-FRET proved
to be fallible.

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# SOEing:

The gBlocks for the VDR-FRET and OHase constructs were both delivered in two separate parts due to production constraints. It was therefore necessary to create the complete VDR-FRET and OHase constructs prior to cloning. The four total delivered parts were respectively termed mCherryVDR, mNeonGreenVDR, OHase1, and OHase2. To facilitate PCR SOEing, they were subjected to PCR amplification.

![Put Image1](https://static.igem.org/mediawiki/2021/9/97/T--Northern_BC--2Results1.png)

Figure 1. PCR Amplification of mCherryVDR, mNeonGreenVDR, OHase1, and OHase2 gBlock constructs

From Figure 1 it is seen that amplification of the gBlocks was successful. The highest bands in the Cherry VDR and the Green VDR correspond roughly to the expected sizes of the gBlocks (1215 bp and 1191 bp respectively). Furthermore, the highest bands in the OHase1 and OHase2 lanes also correspond roughly to their expected band sizes (849 bp and 842 bp respectively). The results from this test indicated that it would be possible to use the products in the syntheses of the next step.

In order to combine the VDR-FRET and OHase sub-pieces, a technique called PCR SOEing was used. Initially, with this technique, mCherryVDR and mNeonGreenVDR were fused together to produce the whole VDR-FRET sequence.

![Put Image2](https://static.igem.org/mediawiki/2021/0/0c/T--Northern_BC--2Results2.png)

Figure 2. PCR SOEing results of the VDR-FRET construct (mNeonGreenVDR + mCherryVDR)

As seen in Figure 2, the highest band observed in the VDR-FRET lane corresponds to roughly 2.5 Kb, which is in concordance with the size of the VDR-FRET construct size (2 426 bp). This suggests that VDR-FRET was successfully synthesized from the mNeonGreenVDR and mCherryVDR gBlocks. Notably, there were extra bands observed at lower size values, indicating that the SOEing of VDR-FRET was not efficient.

Next, the fusion of the OHase1 and OHase2 constructs were tested, again using the PCR SOEing technique, with the goal of creating a complete OHase construct.

![Put Image3](https://static.igem.org/mediawiki/2021/8/8d/T--Northern_BC--2Results3.png)

Figure 3. PCR SOEing results of the OHase construct (OHase1 + OHase2)

In Figure 3 there is a bright band observed at around 1.8 Kb, which corresponds roughly to the size of the OHase construct (1 757 bp). This indicates that there was successful amplification and synthesis of the OHase DNA. Although, similarly to the VDR-FRET SOEing results (Fig. 2), there were extra bands observed.

# Cloning/Transformation:

Following the successful creation of VDR-FRET and OHase, the constructs were ready to be introduced into an *E. coli* system. First, VDR-FRET was ligated into the pSB1C3 plasmid to form a plasmid we named pGEM26 (Fig. 5). This plasmid was then transformed into DH5α E. coli cells, which were grown on selective chloramphenicol media. Colonies that formed were subjected to colony PCR testing using the end primers of the VDR-FRET construct.

![Put Image4](https://static.igem.org/mediawiki/2021/b/ba/T--Northern_BC--2Results4.jpg)

Figure 4. Colony PCR results of transformation of VDR-FRET into pSB1C3.

![Put Image 6](https://static.igem.org/mediawiki/2021/9/92/T--Northern_BC--pGEM26vdrfret.png)
Figure 5. Plasmid map of pGEM26, constructed by the
BioBrick insertion of the VDR-FRET sequence into pSB1C3.

The agarose gel in Figure 4 shows that for each colony selected, following PCR amplification of VDR-FRET, there were observed bands around 2.4 Kb. This suggested that there was successful ligation of VDR-FRET into the pSB1C3 plasmid as well as successful transformation into *E. coli*.

![Put Image 5](https://static.igem.org/mediawiki/2021/e/ea/T--Northern_BC--colonypcr2gel.png)

Figure 6. Colony PCR results of transformation of OHase into pGEM26.

# Sequencing:

From the results in Figure 4, colonies 2.1 and 2.2 were selected to be subject to DNA sequencing in order to further ensure the proper incorporation of the VDR-FRET construct. From the results in Figure 5, the OHase colony was selected to be subject to DNA sequencing in order to ensure the proper incorporation of the OHase construct.

![Put Image6](https://static.igem.org/mediawiki/2021/a/ad/T--Northern_BC--2Results6.PNG)

Figure 8. Sequencing results of the VDR-FRET transformation in colony 2.1

![Put Image7](https://static.igem.org/mediawiki/2021/a/a8/T--Northern_BC--2Results7.PNG)

Figure 9. Sequencing results of the VDR-FRET transformation in Colony 2.2

![put Image 8](https://static.igem.org/mediawiki/2021/d/d7/T--Northern_BC--2Results8.PNG)

Figure 10. Sequencing results of the OHase transformation into pSB1C3\_VDR-FRET

# Assay 1:

After the confirmation of successfully transformed cells, the next step was to measure the fluorescence of our system. To develop a fluorescence baseline, the fluorescence of the system without vitamin D was tested. These tests were performed in triplicate for induced and uninduced *E. coli* BL21 cells as well as untransformed BL21 cells.

![Put Image 9](https://static.igem.org/mediawiki/2021/c/cf/T--Northern_BC--2Results9.PNG)

Figure 11. mNeonGreen fluorescence (RFU) of induced, uninduced, and untransformed BL21 cells as a function of μg of protein from crude lysate, measured with AutoGain.

From Figure 11 it was observed that by increasing the amount of crude lysate, the fluorescence of mNeonGreen after being exposed to light at a wavelength of 485 nm increased proportionally. This was the case for induced cells, uninduced cells, and untransformed BL21 cells. The highest response was found with the uninduced cells expressing VDR-FRET and the lowest was found with the cells that were induced and expressing VDR-FRET.

Next, the fluorescence of mCherry was tested for transformed cell lysates against untransformed BL21 cell lysate by light excitation at a wavelength of 540 nm and emission measurement at a wavelength of 610 nm.

![Put Image 10](https://static.igem.org/mediawiki/2021/6/6d/T--Northern_BC--2Results10.PNG)

Figure 12. mCherry fluorescence (RFU) of induced, uninduced, and untransformed BL21 cells as a function of μg of protein from crude lysate, measured with AutoGain.

From Figure 12 it is seen that both the induced and uninduced cells expressing VDR-FRET displayed an increase in mCherry fluorescence response compared to normal BL21 cells, which did not display any increase. Although, the degree to which the induced and uninduced cells responded with fluorescence is similar.

Lastly, the mNeonGreen/mCherry FRET system as a whole was tested for the uninduced, induced, and untransformed BL21 cells by excitation with light at a wavelength of 485 nm and measuring the emission response at a wavelength of 620 nm, with the addition of increasing amounts of lysates.

![Put Image 11](https://static.igem.org/mediawiki/2021/6/61/T--Northern_BC--2Results11.PNG)

Figure 13. FRET fluorescence (RFU) of induced transformed, uninduced transformed, and untransformed BL21 cells as a function of μg of protein from crude lysate, measured at gain = 110.

From Figure 13 it can be seen that the induced and uninduced cell lysates respond to mNeonGreen excitation wavelengths and respond with mCherry emission wavelengths to high degrees, in comparison to BL21 cell lysate. Similarly to the mCherry testing in Fig. 12, the responses of the induced and uninduced cell lysate product have little variability in comparison to each other.

# Assay 2:

The next step was to determine the range to which certain concentrations of vitamin D affect the observed FRET fluorescence, reflecting on relevant biological levels. The selected concentrations of vitamin D to test were 20 nM and 60 nM

Initially, increasing amounts of induced and uninduced cell lysates had vitamin D added to a final concentration of 20 nM. These reaction volumes were excited to normal FRET excitation (485 nm) and emission (620 nm) wavelengths.

![Put Image 12](https://static.igem.org/mediawiki/2021/0/0a/T--Northern_BC--2Results12.PNG)

Figure 14. FRET fluorescence (RFU) of induced transformed cells at final [1,25(OH)2D] of 20 nM, as a function of μg of protein from crude lysate, measured with AutoGain.

![Put Image 13](https://static.igem.org/mediawiki/2021/c/c7/T--Northern_BC--2Results13.PNG)

Figure 15. FRET fluorescence (RFU) of uninduced transformed cells at final [1,25(OH)2D] of 20 nM, as a function of μg of protein from crude lysate, measured with AutoGain.

Following this, the same conditions of increasing induced and uninduced cell lysate were tested in reaction volumes with final vitamin D concentrations of 60 nM.

![Put Image 14](https://static.igem.org/mediawiki/2021/6/60/T--Northern_BC--2Results14.PNG)

Figure 16. FRET fluorescence (RFU) of induced transformed cells at final [1,25(OH)2D] of 60 nM, as a function of μg of protein from crude lysate, measured with AutoGain.

![Put Image 15](https://static.igem.org/mediawiki/2021/7/7d/T--Northern_BC--2Results15.PNG)

Figure 17. FRET fluorescence (RFU) of uninduced transformed cells at final [1,25(OH)2D] of 60 nM, as a function of μg of protein from crude lysate, measured with AutoGain.

From Figures 14-17 it can be seen that the fluorescence response increases alongside the addition of both 20 nM and 60 nM of vitamin D. Observed in each graph were degrees of variability across the triplicate data. It was found that the least amount of variability in fluorescence response was found when using 60 µg of cell lysate.

# Assay 3:

The next test was to find out if our induced and uninduced cell lysates, as well as if our untransformed BL21 cell lysates, respond with fluorescence to concentrations of vitamin D ranging from 10 nM to 250 nM. Data taken from assay 2 prompted the use of 60 µg of lysate for this test.

![Put Image 14](https://static.igem.org/mediawiki/2021/6/6d/T--Northern_BC--2Results16.PNG)

Figure 18. Relative FRET fluorescence of 60 μg protein from crude lysates of induced, uninduced, and untransformed BL21(DE3) cells with pSB1C3\_VDR-FRET upon addition of 1,25-dihydroxyvitamin D at gain = 110.

From Figure 18 it can be seen that there are slight increases to the fluorescence responses of the induced, uninduced, and BL21 cell lysates. The largest increases in fluorescence are observed with the induced cell lysates, followed by the uninduced cell lysates. The lowest increases in fluorescence were observed with the BL21 cell lysates.

# Protein Purification:

Cell lysates of the VDR-FRET induced, uninduced and untransformed BL21 cells were purified using NEBExpress® Ni Spin Columns (NEB #S1427) and viewed on an SDS-PAGE gel, alongside their unpurified counterparts.

![Put Image 17](https://static.igem.org/mediawiki/2021/f/ff/T--Northern_BC--2Results17.PNG)

Figure 19. SDS-PAGE of crude and purified lysates of induced, uninduced, and untransformed BL21(DE3) strains.

In Figure 19 it is shown that His-Tag purification of VDR-FRET involved the copurification of numerous other proteins. This is shown by the relative distribution of protein bands observed across crude lysate protein as well as purified protein and supernatant. There is a unique band observed in the induced purified supernatant at roughly 80 kDa, similar to the expected band size of VDR-FRET (83 kDa). Similar protein band intensities were observed for each lane, except for the induced purified protein lane, which displayed less overall bands and band intensities.

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