iGEM 2022 — Team uincheon

{% extends "layout.html" %} {% block title %}Notebook{% endblock %} {% block
lead %}Document the dates you worked on your project. This should be a detailed
account of the work done each day for your project.{% endblock %} {% block
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![](https://static.igem.wiki/teams/4310/wiki/team-notebook-banner.png)

[Summary](#link-1)
[Funding Campagin](#link-2)
[Startup field](#link-3)
[The solution](#link-4)
[Key to success](#link-5)
⇪

Detailed records of all experiments

- Week 1 - 2
  Gene Fragments & PCR Primer Dilution
   Week 3 - 4
  TA Cloning of gene fragment for amplification
  BsaI Prefix/Suffix complimentary ssDNA annealing
   Week 4 - 10

  Level 1 SapI Golden Gate Assembly
   Week 8 - 11
  Level 2 BsaI Golden Gate Assembly
   Week 11 - 12
  Level 3 EcoRI/BamHI Double Digestion & pCambia2300 Ligation
   Week 12 -
  Microalgae Transformation

[Link for Detailed Lab Note (in Korean)](https://drive.google.com/file/d/17VYcn-_aA0dk_YvHtTCU2l2XaRe171CA/view?usp=sharing)

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Summary

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Detailed records of experiments

![nb-img](https://static.igem.wiki/teams/4310/wiki/nb-img-1.png)

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{% extends "layout.html" %} {% block title %}Engineering:: {% endblock %} {%
block lead %}Demonstrate engineering success in a part of your project by going
through at least one iteration of the engineering design cycle.{% endblock %} {%
block page\_content %}

![](https://static.igem.wiki/teams/4310/wiki/team-engineering-banner.png)

[Golden Gate Assembly: Prefix/Suffix Block](#link-1)
[General Description](#link-2)
[Detailed Description](#link-3)
[Experiment Design](#link-4)
[Experiment Protocol](#link-5)
[References](#link-6)
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Golden Gate Assembly: Prefix/Suffix Block

For efficient golden gate assembly, our team prepared a gene fragment
that only contains BsaI sequences and its cleavage site so that the
prefix and suffix could be flexibly changed and attached to both ends of
the transcription unit, assembled using SapI. The advantage of this
method is that it is possible to greatly increase the number of gene
fragments that can be synthesized using the golden gate assembly at once
without the need to use various plasmids by ordering a prefix/suffix
fragment at a low price. According to NEB, it was confirmed that their
high-fidelity restriction enzyme could synthesize up to 52 gene
fragments at a time. The first BsaI prefix and the last BsaI suffix have
EcoRI and BamHI cleavage sites for double digestion and ligation to
pCambia 2300 vector for agrobacterium-mediated microalgae
transformation.

[Figure 1] BsaI Prefix/Suffix

![fr-img](https://static.igem.wiki/teams/4310/wiki/engi-img-1.png)

![fr-img](https://static.igem.wiki/teams/4310/wiki/engi-img-2.png)

![fr-img](https://static.igem.wiki/teams/4310/wiki/engi-img-3.png)

![fr-img](https://static.igem.wiki/teams/4310/wiki/engi-img-4.png)

[Figure 2] Simple Diagram of level 1~3 assembly

![fr-img](https://static.igem.wiki/teams/4310/wiki/engi-img-5.png)

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{% extends "layout.html" %} {% block title %}Experiments:: {% endblock %} {%
block lead %}Describe the research, experiments, and protocols you used in your
iGEM project.{% endblock %} {% block page\_content %}

![](https://static.igem.wiki/teams/4310/wiki/team-experiments-banner.png)

Experiment Design

*Biosensor #1: Heat shock-inducible Promoter*

Heat shock-inducible promoter activity in C. vulgaris is tested by
observing GFP expression.
The fluorescence intensity was compared
with control.

- pHsp70A > Kozak > egfp > Tnos
  a. Incubation in the dark and shifted into the light for 1 h.
  b. Incubation in the light and exposed to heat shock (40°C) for 30
  min.p35S > Kozak > eGFP > Tnos (Control)

*Biosensor #1-2: Cold-inducible Promoter*

Cold-inducible promoter activity in C. vulgaris is tested by observing
GFP expression.
The fluorescence intensity was compared with
control.

- pCnAFP>Kozak>eGFP>Tnos
  a. Incubation in low temperature (10°C) for 2 hour.p35S>Kozak>eGFP>Tnos (Control)

*Biosensor #2: Bioswitch: Inducible Promoter - Recombinase
Activity*

Recombinase activity, triggered by inducible promoters, is tested by
observing GFP expression.
The fluorescence intensity was compared
with control.

p35S>attB>mCherry>Tnos : pHSP70A>Kozak>phiC31>Tnos :
attP>Kozak>eGFP>Tnos

(Heat shock inducible promoter + phiC31 recombinase)

- - After recombination : p35S > attB-attP > Kozak > eGFP > Tnos
  a. Incubation in the dark and shifted into the light for 1 h.

  b. Incubation in the light and exposed to heat shock (40°C) for 30
  min.

  p35S>attB>mCherry>Tnos : pCnAFP>Kozak>phiC31>Tnos :
  attP>Kozak>eGFP>Tnos
  (Cold inducible promoter + phiC31 recombinase)
  - After recombination : p35S > attB-attP > Kozak > eGFP > Tnos
  a. Incubation in low temperature (10°C) for 2 hours.

  p35S>loxP(1)>mCherry>Tnos : pHSP70A>Kozak>Cre>Tnos :
  loxP(2)>Kozak>eGFP>Tnos
  (Heat shock inducible promoter + Cre recombinase)
  - After recombination : p35S > loxP(1)-loxP(2) > Kozak > eGFP > Tnos

  a. Incubation in the dark and shifted into the light for 1 h.

  b. Incubation in the light and exposed to heat shock (40°C) for 30
  min.

  p35S>loxP(1)>mCherry>Tnos : pCnAFP>Kozak>Cre>Tnos :
  loxP(2)>Kozak>eGFP>Tnos

  (Cold inducible promoter + Cre recombinase)
  - After recombination : p35S > loxP(1)-loxP(2) > Kozak > eGFP > Tnos

  a. Incubation in low temperature (10°C) for 2 hours.p35S>eGFP>nos terminator (Control)

*Biosensor #3: Quorum-Sensing Promoter & Protein*

Quorum-Sensing promoter activity in C. vulgaris is tested by observing
GFP expression.
The expression is observed in various
concentrations of N-3-oxo-octanoyl homoserine lactone (C8 AHL).
The observation is made in liquid medium with 0 μM, 0.1 μM, 1 μM, 100μM,
1000 μM, 10000 μM C8 AHL. The fluorescence intensity was compared with
control.

p4X(TraR)::m35S>Kozak>eGFP>Tnos : pHSP70A/pRbcS2>Kozak>VP16
TAD::TraR>Tnos

p4X(TraR)::m35S>Kozak>eGFP>Tnos : p35S>Kozak>VP16 TAD::TraR>Tnos

p35S>Kozak>eGFP>Tnos (Control)

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Experiment Protocol

Microalgae Transformation (Cha et al., 2012)

1. The mediums and supplements for microalgae transformation are
   obtained from Kisan Bio, South Korea.

1. 1. Supplement Luria Broth with 5 mM glucose, 50 mg/L kanamycin
2. 2. Inoculate Agrobacterium. Tumefaciens (KCTC 12031) single colonies
   in 10mL of Luria Broth
3. 3. Under constant agitation at 200 rpm on a rotary shaker at 30°C,
   culture overnight in the dark
4. 4. Inoculate 5mL of overnight culture from step 3 in 50mL of same
   medium
5. 5. Under constant agitation at 200 rpm at 27°C, culture until OD600
   = 0.8–1.2 in the dark
6. 6. Centrifuge to harvest bacterial culture
7. 7. Wash with induction medium (BG11 + 100 μM acetosyringone, pH 5.6)
   and dilute to OD600 = 0.5
8. 8. Pre-culture total of 5 × 106 Chlorella cells from a log-phase
   culture (OD600 = 0.5–1.0) on BG11 solidified with 1.5% (w/v)
   bacto-agar at 25°C for 5 days
9. 9. Harvest bacterial culture with induction medium
10. 10. Mix algal pellet with 200 μL bacterial suspension
11. 11. Plate on induction medium solidified with 1.5% (w/v) bacto-agar
12. 12. Co-cultivate algae with bacteria (3 days, in dark, 25°C)
13. 13. Harvest cells with 7mL of BG11 supplemented with 500 mg/L
    cefotaxime and 50 mg/L kanamycin to eliminate Agrobacterium (2 days,
    in dark, 25°C)
14. 14. Measure eGFP expression
15. 15. Plate the remaining cells on selective media with 50 mg/L
    kanamycin and 500 mg/L cefotaxime
16. 16. Incubate transformed cells in dark before exposure to light (2
    days, 25°C)
17. 17. Grow cells on LB agar plates to detect any Agrobacterium (7 days
    in dark, 25°C)

Fluorescence microplate reader assay (Molino et al., 2018)

1. 1. Inoculate transformed colony in 1mL BG11 selective media and
   incubate at 25°C under constant illumination (50 μmol photons/m2/s)
   and constant agitation at 100–150 rpm on a rotary shaker, culture
   for 7 days
2. 2. Transfer 100 μL cells to wells of a black, clear bottom 96-well
   plate
3. 3. Set up microplate reader (ThermoFisher Scientific, USA) as table
   1
4. 4. Measure fluorescence using BG11 medium as blank and wild type
   cells as negative control
5. 5. Repeat steps 5-7 with supernatant in the Deep-well plate obtained
   by centrifugation at 3000 ×g for 10 min
6. 6. The fluorescent signal is normalized by OD600.

![fr-img](https://static.igem.wiki/teams/4310/wiki/engi-img-6.png)

Fluorescence microscopic observation of EGFP expression
(Yang et al., 2015)

1. The mediums and supplements for microalgae transformation are
   obtained from Kisan Bio, South Korea.

1. - eGFP fluorescence was visualized by using a narrow-band filter
   (Olympus U-FBNA, excitation filter 470–495 nm, barrier filter
   510–550 nm).
2. - Chlorophyll autofluorescence was observed using a wide-band filter
   (Olympus U-FBW, excitation filter 460–495 nm, barrier filter 510 nm)

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{% extends "layout.html" %}
{% block title %}Results{% endblock %}
{% block lead %}You can describe the results of your project and your future plans here.{% endblock %}
{% block page\_content %}

## What should this page contain?

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* Clearly and objectively describe the results of your work.
* Future plans for the project.
* Considerations for replicating the experiments.

## Describe what your results mean

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* Interpretation of the results obtained during your project. Don't just show a plot/figure/graph/other, tell us what you think the data means. This is an important part of your project that the judges will look for.
* Show data, but remember **all measurement and characterization data must also be on the Part's Main Page on the [Registry](http://parts.igem.org/Main_Page).** Otherwise these data will not be in consideration for any medals or part awards!
* Consider including an analysis summary section to discuss what your results mean. Judges like to read what you think your data means, beyond all the data you have acquired during your project.

## Project Achievements

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You can also include a list of bullet points (and links) of the successes and failures you have had over your summer. It is a quick reference page for the judges to see what you achieved during your summer.

* A list of linked bullet points of the successful results during your project
* A list of linked bullet points of the unsuccessful results during your project. This is about being scientifically honest. If you worked on an area for a long time with no success, tell us so we know where you put your effort.

## Inspirations

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* [2019 Newcastle](https://2019.igem.org/Team%3ANewcastle/Results)
* [2019 Munich](https://2019.igem.org/Team%3AMunich/Results)
* [2019 Tec Chihuahua](https://2019.igem.org/Team%3ATec-Chihuahua/Results)
* [2020 Aalto Helsinki](https://2020.igem.org/Team%3AAalto-Helsinki/Results)
* [2020 GreatBay SCIE](https://2020.igem.org/Team%3AGreatBay_SCIE/Results)
* [2020 Queens Canada](https://2020.igem.org/Team%3AQueens_Canada/Results)

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