iGEM 2023 — Team georgiastate-swjtu

{% extends "layout.html" %}
{% block title %}Notebook{% endblock %}
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## Notebook Documents

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##### Ethanol Team Notebook

##### Knockout Team Notebook

##### PHB Team Notebook (Part I)

##### PHB Team Notebook (Part II)

## Inspirations

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* [Ethanol Team Notebook](#pdf1)
* [Knockout Team Notebook](#pdf2)
* [PHB Team Notebook (Part I)](#pdf3)
* [PHB Team Notebook (Part II)](#pdf3)

## What should this page contain?

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* Include pictures of your teammates, don't forget instructors and advisors!
* You can add a small biography or a few words from each team member, to tell us what you like, and what motivated you to participate in iGEM.
* Take team pictures! Show us your school, your lab and little bit of your city.
* Remember that image galleries can help you showcase many pictures while saving space.

**Important:** Your wiki pages will be archived at the end of the iGEM season and this content will remain online. Please keep this in mind as you post photos and personal information on this page.

## Inspirations

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* [2019 CU](https://2019.igem.org/Team%3ACU/Team)
* [2019 UANL](https://2019.igem.org/Team%3AUANL/Team)
* [2019 William and Mary](https://2019.igem.org/Team%3AWilliam_and_Mary/Team)
* [2020 BOKU Vienna](https://2020.igem.org/Team%3ABOKU-Vienna/Team)
* [2020 CAU China](https://2020.igem.org/Team%3ACAU_China/Team_Member)
* [2020 Lethbridge](https://2020.igem.org/Team%3ALethbridge/Members)

Names of each student: Yuqi Song, Xiang Li(Alice), Chuqiang Peng
Date: 9.27.23
Protocols Performed: Overnight culture E.coli JM109 in liquid LB medium.
Purpose: To revive dormant E. coli and make more E.coli for the next step.
Supplies used/Materials: E.coli JM109, liquid LB medium with AMP
Results:
Conclusions/Next Lab goals: Make solid LB medium and overnight E.coli on it.
Extra notes:
Names of each student: Yuqi Song, Xiang Li(Alice), Chuqiang Peng
Date: 9.28.23
Protocols Performed: Overnight culture E.coli on LB plate
Purpose: Prepare for next step
Supplies used/Materials: Solid LB plate , E.coli
Results:
Conclusions/Next Lab goals: Culture E.coli in liquid LB
Extra notes:
Names of each student: Xiang Li(Alice)
Date: 9.29.23
Protocols Performed: Culture E.coli in liquid LB
Purpose: prepare for next step
Supplies used/Materials: Liquid LB medium, E.coli
Results:
Conclusions/Next Lab goals: Culture E.coli in liquid LB with ethanol
Extra notes:
Names of each student: Chuqiang Peng
Date: 9.30.23
Protocols Performed: 100μL culture was inoculated into a 10-ml screw-cap tube filled with 9.9 ml LB liquid medium and added 10μL ethanol. Overnight culture.
Purpose: Increase the ethanol tolerance of E.coli
Supplies used/Materials: 95%ethanol, E.coli, LB
Results:
Conclusions/Next Lab goals: Dilute the solution
Extra notes:
Names of each student: Yuqi Song
Date: 10.1.23
Protocols Performed: 100μL culture was inoculated into a 10-ml screw-cap tube filled with 9.9 ml LB liquid medium and added 10μL ethanol. Overnight culture.
Purpose: Increase the ethanol tolerance of E.coli
Supplies used/Materials: 95%ethanol, E.coli, LB
Results:
Conclusions/Next Lab goals: overnight culture E.coli in LB plate.
Extra notes:Names of each student: Kaia and Jasmine
Date: 9/25/2023
Protocols Performed:
Located the E.coli, JM109. Made LB agar and broth
LB Broth:
Weigh 5g peptone, 2g yeast, and 5g NaCl into a weigh boat.
Pour the powders into a glass bottle containing 500mL of diH2O.
Mix the mixture and label it “Knockout | iGEM | 7-12-2023 LB broth.”
Agar:
Weigh 5g peptone, 2g yeast, 5g NaCl, and 5g agar onto weigh boat.
Add the powders into a glass jar containing 500mL of diH2O.
Mix the mixture. Do NOT add antibiotic (ampicillin) yet.
Label it “ knockout | iGEM | 7-12-2023 Agar without Ampicillin.”
Place the two glass bottles into the autoclave for 30 min.
Purpose:
The LB agar will be used to culture the E.coli and will also be used to plate a few.
Supplies used/Materials:
Peptone, yeast, NaCl, weigh boat, agar, diH2O, 500mL glass bottle, autoclave.
Results:
N/A
Conclusions/Next Lab goals:
Pour the LB agar into the plates.
Extra notes:
A group of bottles with liquid in them
Description automatically generated
Names of each student: Tracy
Date: 9/26/2023
Protocols Performed:
Pouring LB into plate.
Completely melt the LB agar on a hot plate.
Place the plates upside down before pouring.
Keep an open flame and place the plates and agar close to it. Do not open the plates until pouring time.
Keeping close to the flame, use one hand to first run the agar bottle through the flame and the other hand to carefully open the culture plate’s lid. Pour until the agar is covering all the areas of the plate with no air bubbles.
Repeat step 4 sixteen more times. Stay close and below the flame.
Label the stack “iGEM Fall 2023 No Amp. KO 9/26/2023.
Store in refrigerator for the next use.
Purpose:
To prep the culture experiment by pouring the LB agar that was previously made into the culture plates with no ampicillin.
Supplies used/Materials:
LB Agar, hot plate, open flame, lighter, 17 culture plates.
Results:
N/A
Conclusions/Next Lab goals:
Get started with SOC prep.
Extra notes:
Names of each student: Kaia and Jasmine
Date: 9/27/2023
Protocols Performed:
SOC media
First prepare the solutions：
Per liter: To 950 mL of deionized H20, add:
Tryptone 20g
Yeast extract 5g
NaCI 0.5g
250 mM solution of KCI.
KCI：1.86g
Deionized H2O：100 mL
2 M MgCl2.
MgCl2: 19g
Deionized H2O：90ML
1 M solution of glucose.
Glucose：18 g
Deionized H2O：90ML
Adjust the PH of all solutions to 7.0 and then sterilize all solutions by autoclaving for 20 min at 15 psi (1.05 kg/cm2) on liquid cycle. Just before use, add 10 ml of a sterile solution of 250 mM KCl, 5 mL of a sterile solution of 2 M MgCl2 and 20 mL of a sterile 1 M solution of glucose.
Purpose:
SOC will be used run the plasmid and bacteria transformation.
Supplies used/Materials:
Tryptone, yeast, NaCl, KCl, MgCl2, glucose, diH2O, pH meter, autoclave
Results:
Conclusions/Next Lab goals:
Begin heat shock.
Extra notes:
Names of each student: Nancy
Date: 9/28/2023
Protocols Performed:
Electroporation trial run
Purpose:
To test that the electroporation would be successful, a trial test was ran with a different E.coli strain.
Supplies used/Materials:
Results:
Conclusions/Next Lab goals:
Heatshock.
Extra notes:
Names of each student: Kaia and Jasmine
Subteam name: Knockout
Date: 10/4/2023
Protocols Performed:
Heatshock
Thaw chemically competent bacterial cell such as JM109 on ice.
Combine 40 uL of JM109 with 1uL (1ng/ uL) of plasmid to 1.5mL eppendorf tube.
Incubate cells on ice for 35 minutes.
After ice incubation, place the samples into 42℃ water bath for 4 seconds.
Put the samples back to ice for 2 minutes to recover.
Quickly take the eppendorf tube out and immediately add 250uL of SOC medium.
Place the eppendorf tube into 37℃ shaking water incubator for 1hr and 2omin at 250rpm.
After shaking, streak 150uL of solution onto an LB agar plate with the respective antibiotic (ampicillin).
Purpose:
To separate the plasmid from E.coli
Supplies used/Materials:
Ice bath, eppendorf tube, ampicillin, SOC, shaking incubator, JM109, LB agar plate.
Results:
Conclusions/Next Lab goals:
Extra notes:
Names of each student: Elizabeth Plumart
Date: 10.07.23
Protocols Performed: Transformation (heat shock)
Purpose: Transform recET and ackA knockout into E. coli
Results: Success
Conclusions/Next Lab goals: Overnight culture for mini-prep
Extra notes:
Names of each student: Elizabeth Plumart
Date: 09.18.23
Protocols Performed: Heat shock
Purpose: Transform phaB, phaC, pChlamdy into E. coli
Results: Success
Conclusions/Next Lab goals: Overnight culture to make glycerol stocks, transform phaA, tdTomato, algae promoter, algae terminator into E. coli
Extra notes:
Names of each student: Elizabeth Plumart, Chloe Benjamin
Date: 09.22
Protocols Performed: Overnight culture
Purpose: Grow up colonies from transformation (phaB, phaC, pChlamdy)
Results: Success
Conclusions/Next Lab goals: Mini-prep
Extra notes:
Names of each student: Elizabeth Plumart
Subteam name: Algae PHB
Date: 09.23
Protocols Performed: Mini-prep
Purpose: Extract plasmid (phaB, phaC, pChlamdy) from overnight culture
Results: Success
Plasmid/Gene, Colony
Concentration (ng/ul)
260/280
pChlamdy, 1
47.8
1.92
pChlamdy, 2
56.4
1.92
pChlamdy, 3
48.7
1.93
phaC, 1
37.9
1.86
phaC, 2
51.5
1.90
phaC, 3
64.4
1.94
phaB, 1
505.7 (measured 3 times)
1.85 (260 = 10.114, 280 = 5.464)
phaB, 2
48.3
1.93
phaB, 3
48.6
1.91
Conclusions/Next Lab goals: Transform kit plate parts, restriction digest & ligation to build expression construct
Extra notes:
Names of each student: Elizabeth Plumart, Chloe Benjamin
Date: 09.25.23
Protocols Performed: Heat shock
Purpose: Transform phaA, CaMV 35S, 3’ UTR, mCherry into E. coli
Results: Success
Conclusions/Next Lab goals: Overnight culture to make glycerol stocks, colony PCR on kit plate parts
Extra notes:
Names of each student: Chloe Benjamin
Date: 09.26.23
Protocols Performed: overnight culture of E.coli plates (phaA, mCherry, CaMV 35S, 3’ UTR)
Purpose: to prepare for the Miniprep and making glycerol stocks tomorrow
Results: Success
Conclusions/Next Lab goals: Colony PCR, Miniprep, Glycerol stocks, Restriction Digest to create algae construct, Ligation, Transform algae construct into E.coli
Extra notes: It was decided that the colony pcr will be done on Wednesday, 9/27/23 instead. The shaker in nsc 318 was not working last night, so I put the overnight culture tubes in another incubator in nsc 318. The E.Coli plates were placed back in the fridge in nsc 110 and the purple box containing antibiotics were placed in the freezer in nsc 110.
Names of each student: Xucheng Zhang
Date: 09.27.23
Protocols Performed: colony pcr and gel electrophoresis
Purpose: confirms E.coli has the genes mCherry (reporter gene), CaMV 35s(promoter), and 3’ UTR (terminator)
Results: Success
Conclusions/Next Lab goals: Miniprep
Extra notes:
Names of each student: Chloe Benjamin, Elizabeth Plumart
Date: 09.27.23
Protocols Performed: miniprep
Results: Success
Plasmid/Gene, Colony
Concentration (ng/ul)
260/280
phaA 1
80.4
1.92
phaA 2
96.8
1.93
phaA 3
90.3
1.92
CaMV 1
52.5
1.98
CaMV 2
57.1
1.98
CaMV 3
71.1
1.92
3’ UTR 1
71.5
1.95
3’ UTR 2
62.3
1.95
3’ UTR 3
57.3
1.96
mCherry 1
46.6
2.01
mCherry 2
65.3
1.99
mCherry 3
61.6
1.99
Conclusions/Next Lab goals: Miniprep
Extra notes:
Names of each student: John Biggs
Date: 09.28.23
Protocols Performed: Restriction digest
Restriction digest enzymes
Gene
Enzymes
CaMV 35S (algae promoter)
SpeI & PstI
mCherry (reporter)
XbaI & PstI
phaA
XbaI & PstI
phaB
XbaI & PstI
phaC
XbaI & PstI
Purpose: Prepare construct for expression
Results: phaA, phaB, phaC success, CaMV35S, mCherry fail
Conclusions/Next Lab goals:
The next step is completing the remaining restriction digests, gel extractions, and ligations to finish building the construct we want to express.
Extra notes:
Names of each student: Chloe Benjamin, Elizabeth Plumart
Date: 10/2/23
Protocols Performed:
Gel electrophoresis to verify if the restriction digests completed on 9/28/23 were done successfully
Restriction digest enzymes
Gene
Enzymes
CaMV 35S (algae promoter)
SpeI & PstI
mCherry (reporter)
XbaI & PstI
phaA
XbaI & PstI
phaB
XbaI & PstI
phaC
XbaI & PstI
Run out on gel, gel extraction on band.
Purpose: to prepare for construct for expression
Supplies used/Materials:
Results: The DNA bands did not travel far enough down the gel, so we did not get successful results to analyze.
Conclusions/Next Lab goals: The next goal is to repeat the restriction digests on 9/28/23.
Extra notes:
Names of each student: Xucheng Zhang
Date: 10/3/23
Protocol performed: Restriction digests, gel electrophoresis, and gel extraction
Restriction digest enzymes
Gene
Enzymes
CaMV 35S (algae promoter)
SpeI & PstI
mCherry (reporter)
XbaI & PstI
phaA
XbaI & PstI
phaB
XbaI & PstI
phaC
XbaI & PstI
Gel extraction of each after gel electrophoresis should be done
Purpose: to prepare for the expression of a PHB construct
Supplies used/Material:
Results:
From left to right, the genes are in the following order starting at the 2nd well since the first well has the DNA ladder: CaMV 35S (promoter gene), mCherry (reporter gene),
Conclusion/Next lab goals:
Restriction digests of phAa and phAB were a success as seen in the gel electrophoresis results. However, restriction digests of the other genes will be redone.
Extra notes:
Names of each student:
Chloe Benjamin
Date: 10/3/23
Protocol performed: overnight culture to grow E.coli bacteria containing the genes, phAc and mcherry
Purpose: to prepare for a miniprep as well prepare for the expression of a PHB construct
Results:
Conclusion/Next lab goals:
Restriction digests
Gel extraction
Ligation
Miniprep to isolate the plasmid containing the genes mcherry and phAc
Names of each student:
Xucheng Zhang
Chloe Benjamin
Elizabeth Plumart
Date: 10/4/23
Protocol performed:
Restriction digests and Gel extraction
Ligation of phAa and phAb
Miniprep to isolate plasmid containing the genes mcherry and phAc ~This will only be done if Eric cannot complete a gel extraction of mcherry and phAc due to DNA bands for both genes not being present on the gel.
Restriction enzymes
Gene
Enzymes
CaMV 35S (algae promoter)
SpeI & PstI
mCherry (reporter)
XbaI & PstI
phaA
XbaI & PstI
phaB
XbaI & PstI
phaC
XbaI & PstI
Ligate each CDS to 3’UTR.
Purpose: to prepare for the expression of a PHB construct
Results: mCherry, CA MV 35S, and terminator failed
Conclusion/Next lab goals: Miniprep
Names of each student: Xucheng
Subteam name: Algae PHB
Date: 10/5/23
Protocol performed:
Measured results of mini prep completed on 10/4/23
Miniprep to isolate the plasmids containing the promoter and terminator
Results:
Promoter Results:
P1 = Concentration: 3.8 ng/microliter and 260/280: 3.61
P2= Concentration: 7.4 ng/microliter and 260/280: 2.92
P3= Concentration: 15.3 ng/microliter and 260/280: 2.10
P4= Concentration: 4.0 ng/microliter and 260/280: 3.20
Terminator Results:
T1= Concentration: 24.1 ng/microliter and 260/280: 2.06
T2=Concentration: 39.7 ng/microliter and 260/280: 1.67
T3=Concentration: 2.5 ng/microliter and 260/280: 3.77
T4=Concentration: 7.7 ng/microliter and 260/280: 2.10
phAc Results and mCherry Results
Plasmid Gene,Colony
Concentration
260/280
1-phaC 1
97 nanograms/microliter
1.87
1-phaC 2
57.6 nanograms/microliter
1.87
1-phaC 3
47.2 nanograms/microliter
1.84
2-phaC 1
91.1 nanograms/microliter
1.82
2-phaC 2
70.0 nanograms/microliter
1.94
Plasmid Gene,Colony
Concentration
260/280
2-phaC 3:
78.5 nanograms/microliter
1.87
1-mcherry 1:
9.4 nanograms/microliter
1.71
1-mcherry 2:
8.1 nanograms/microliter
1.64
1-mcherry 3:
8.7 nanograms/microliter
1.77
Plasmid Gene,Colony
Concentration
260/280
2-mCherry 1:
8.7 nanograms/microliter
1.58
2-mCherry 2:
7.9 nanograms/microliter
1.64
2-mCherry 3:
6.6 nanograms/microliter
1.63
Names of each student:
Elizabeth Plumart
Subteam name: Algae PHB
Date: 10.07.23
Protocol performed:
Transformation of mCherry, CA MV 35S, and terminator
Results:
Terminator failed (one colony that we can try), CA MV 35S and mCherry success
Names of each student: Elizabeth Plumart, Chloe Benjamin
Date: 10/09/23
Protocols Performed:
Mini prep
Restriction digest
Restriction digest enzymes
Gene
Enzymes
CaMV 35S (algae promoter)
SpeI & PstI
mCherry (reporter)
XbaI & PstI
Terminator
EcoRI & XbaI
Results:
Mini prep- Success
Plasmid/Gene, Colony
Concentration (ng/ul)
260/280
CAMV 35S, 1
142.3
1.95
CAMV 35S, 2
122.3
1.94
mCherry, 1
135.4
1.92
mCherry, 2
131.5
1.92
terminator
128.2
1.92
Restriction digest- Success
Names of each student: Xucheng Zhang, Chloe Benjamin
Date: 10/10/23
Protocols Performed: Gel extraction, ligation, transformation
Results:
Gel extraction- success
Ligation- success
Transfo
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{% extends "layout.html" %}
{% block title %}Engineering Success{% 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 %}

Engineering Design-Build-Test-Learn
Bio-safety System Design
Parts

# Engineering Design-Build-Test-Learn

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### Our ultimate goal is to produce the bioplastic PHB…

…but how? Where do we start?

Like all engineering, Bio-Barbie uses scientific principles to engineer biological tools to synthesize bioplastic.

## Overview

![Image](static/assets/Project_Overview-min.jpeg)

Polyhydroxybutyrate (PHB) is one of the most well-studied bioplastics. PHB is a common type of polyhydroxyalkanoate (PHA), a class of molecules produced for energy storage when the microorganism is under stress. Our chassis for PHB production, C. reinhardtii, naturally makes a low amount of PHB under such stressful conditions, such as nutrient starvation (Cassuriaga et al. 2020). In co-culture with E. coli, we look to push PHB production even further (Yamada et al. 2023).

Our project increases PHB production at the beginning, middle, and end of PHB synthesis. We accomplish this by:

1. Genetically modifying C. reinhardtii to express phaA, phaB, and phaC to increase PHB production
2. Inducing stress in C. reinhardtii with an E. coli strain genetically modified to produce ethanol, a stress factor
3. Inactivating ackA-pta to increase acetyl-CoA available for PHB synthesis

## PHB Synthesis in C. reinhardtii

To increase the amount of PHB produced by C. reinhardtii, we designed three genes: phaA (BBa\_K4736005), phaB (BBa\_K4736006), and phaC (BBa\_K4736007). These genes come from Cupriavidus necator, a soil bacterium previously known as Ralstonia eutropha. We selected these genes because C. necator is able to accumulate PHB up to 90% of its cell dry weight (Obruca et al., 2014). After modifying these genes to be compatible with the RFC 10 standard, they were synthesized by Integrated DNA Technologies (IDT).

To build our construct for expression, we selected the promoter CaMV35S (BBa\_J428074) and eukaryotic terminator (BBa\_J428081) from the Plants Collection in the 2023 iGEM Kit Plate.

Since our lab has never successfully transformed a microalgae, we also designed an expression construct for mCherry (BBa\_K4736008), a reporter gene that is compatible with C. reinhardtii, using the same promoter and terminator. This allows us to easily and quickly screen our algae culture for successful transformants.

We have successfully built these constructs through a series of restriction digests, gel extractions, ligations, transformations, and mini-preps, and we have transformed them into E. coli to test if our PHB genes are functioning as expected. We expect to have completed at least one round of testing by the 2023 iGEM Jamboree. Once we have confirmed that our genes are working as expected, we will transform them into C. reinhardtii and test for increased PHB production. Based on these results, we may modify our design or experiment with different promoters and culture conditions to maximize PHB production.

## Inducing Stress with Ethanol

Ethanol can be used to induce exogenous stress, resulting in excessive production of PHB (Obruca et al. 2010). Besides its role as a stress factor, ethanol can also be directly converted into acetyl-CoA, the starting point in PHB production (Sun et al. 2020). Finally, ethanol automatically flows out of cell through overflow metabolism, avoiding the need to design a pump or destroy any cells (Paczia et al. 2012).

We’ve designed two genes that work together to produce ethanol: pdc (BBa\_K4736000) and adhB (BBa\_K4736001), both from the bacteria Zymomonas mobilis. These two genes create a pathway, which uses pyruvate to produce ethanol (Wang et al. 2008). We’ve also designed a composite part to express both of these genes (BBa\_K4736002).

Currently, we are culturing E. coli in an environment with increasing ethanol concentration to screen out ethanol-tolerant E. coli (Wang et al. 2008). Then, we will transform the two ethanol-producing genes into our ethanol-tolerant E. coli strain. We will measure the tolerance of algae to determine the ideal concentration of ethanol. The final step will be to co-culture the modified E. coli and C. reinhardtii and measure for increased PHB production.

## Acetate Knockout for Increased Acetyl-CoA

In order to increase the amount of acetyl-CoA available for PHB synthesis, we’ve designed two genes to redirect acetyl-CoA flux away from acetate production (Ku et al. 2020). These genes will knockout the ackA-pta gene, which is the main pathway for acetate production in E. coli. We expect that this knockout will create an excess of acetyl-CoA that in turn will be converted to citrate through the TCA cycle and leave the cell through overflow metabolism. In a co-culture setting, this citrate would be transferred to C. reinhardtii, where it can be used starting point for PHB synthesis.

We are using a gene inactivation method developed by Datsenko and Wanner, in which short regions of homology, created through PCR, are used to disrupt target genes (Datsenko and Wanner 2000). However, using regions homologous to the ackA-pta gene that were previously created by Centeno-Leija et al., we were able to order our knockout construct in once piece from IDT (Centeno-Leija et al. 2014). The construct (BBa\_K4736004) consists of a gene for ampicillin resistance, flanked by two short regions homologous to the ackAF and ptaR genes, respectively.

In order to facilitate the recombination of our DNA construct into E. coli’s genome, we designed the gene recET. It codes for the enzyme exonuclease VIII, which can facilitate homologous recombination of linear DNA between short regions of homology. It is analogous to lambda Red recombinase, the enzyme used by Datsenko and Wanner.

## References:

* Cassuriaga, A., et al. (2020). *Polyhydroxybutyrate production and increased macromolecule content in Chlamydomonas reinhardtii cultivated with xylose and reduced nitrogen levels.* International Journal of Biological Macromolecules. <https://doi.org/10.1016/j.ijbiomac.2020.04.273>
* Datsenko, K. A., Wanner, B. L. (2000). *One-step Inactivation of Chromosomal Genes in Escherichia Coli K-12 Using PCR Products.* Proceedings of the National Academy of Sciences of the United States of America. <https://doi.org/10.1073/pnas.120163297>
* Ku, J. T., et al. (2020). *Metabolic Engineering Design Strategies for Increasing Acetyl-CoA Flux.* Metabolites vol. 10,4 166. 23 Apr. 2020, <https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7240943/>
* Obruca, S., Benesova, P., Oborna, J. et al. (2014). *Application of protease-hydrolyzed whey as a complex nitrogen source to increase poly(3-hydroxybutyrate) production from oils by Cupriavidus necator.* Biotechnol Lett 36, 775–781. <https://doi.org/10.1007/s10529-013-1407-z>
* Obruca S, Marova I, Stankova M, Mravcova L, Svoboda Z. (2010). *Effect of ethanol and hydrogen peroxide on poly(3-hydroxybutyrate) biosynthetic pathway in Cupriavidus necator H16.* World J Microbiol Biotechnol. <https://pubmed.ncbi.nlm.nih.gov/24026931/>
* Paczia N, Nilgen A, Lehmann T, Gätgens J, Wiechert W, Noack S. (2012). *Extensive exometabolome analysis reveals extended overflow metabolism in various microorganisms.* Microb Cell Fact. <https://pubmed.ncbi.nlm.nih.gov/22963408/>
* Sun S, Ding Y, Liu M, Xian M, Zhao G. (2020). *Comparison of Glucose, Acetate and Ethanol as Carbon Resource for Production of Poly(3-Hydroxybutyrate) and Other Acetyl-CoA Derivatives.* Front Bioeng Biotechnol. <https://pubmed.ncbi.nlm.nih.gov/24026931/>
* Wang, Z., Chen M., Xu, Y. et al. (2008). *An ethanol-tolerant recombinant Escherichia coli expressing Zymomonas mobilis pdc and adhB genes for enhanced ethanol production from xylose.* Biotechnol Lett. <https://doi.org/10.1007/s10529-007-9597-x>
* Yamada, R., Yokota, M., Matsumoto, T., Hankamer, B., Ogino, H. (2023). *Promoting cell growth and characterizing partial symbiotic relationships in the co-cultivation of green alga Chlamydomonas reinhardtii and Escherichia coli.* Biotechnology Journal. <https://pubmed.ncbi.nlm.nih.gov/36479591/>

# Biosafety System Design

### Introduction:

The increasing applications of synthetic biology emphasize the requirement for robust biosafety measures. Our project exploits the symbiotic co-culturing of E. coli and C. reinhardtii. To ensure that these genetically modified organisms don't pose environmental threats upon potential release, we have implemented a two-fold biosafety strategy involving a toxin-antitoxin system in E. coli and an auxotrophy mechanism in C. reinhardtii.

#### E. coli Kill Switch Design using the Toxin-Antitoxin System: **1. Gene Selection:** **Toxin:** RelE, a ribonuclease, that upon expression, inhibits translation by cleaving mRNAs in the ribosomal A-site. **Antitoxin:** RelB, which binds and neutralizes the toxic effect of RelE, produced at a high level when induced. **2. Promoter, Regulator & Inducer Selection for Antitoxin Production:** **Promoter for Antitoxin:** Ptrc promoter – A hybrid of trc and lac promoters. This promoter is renowned for its tight regulatory control and is often used in IGEM, offering low basal expression levels in the absence of an inducer, ensuring that the antitoxin doesn't unnecessarily burden the bacterial metabolism. However, upon the introduction of the inducer IPTG, the Ptrc promoter provides strong and robust expression, ensuring a sufficient amount of RelB antitoxin is produced to counteract the toxin. **Regulator:** The LacI protein plays a pivotal role in the functionality of the Ptrc promoter. Without the inducer, LacI binds to Ptrc, preventing transcription. This regulation ensures that the antitoxin isn't produced in environments where it's not needed. **Inducer:** IPTG is used as the synthetic inducer. When introduced into the environment, IPTG binds to LacI, causing a conformational change that prevents it from binding to the Ptrc promoter. This, in turn, induces the transcription of the downstream genes, in this case, the RelB antitoxin. **3. Promoter for Toxin Production:** **Promoter for Toxin:** PJ23101 promoter - This is a well-established constitutive promoter widely used in synthetic biology for E. coli applications. Being constitutive, it ensures a steady and consistent level of gene expression without the requirement of any inducers. By employing the PJ23101 promoter to control the expression of the toxin gene, we're ensuring a continual, moderate level production of the toxin. This level is counteracted by the high expression of antitoxin when in the laboratory condition with the inducer present. However, in the event of an escape, the reduced expression of antitoxin combined with the consistent production of toxin ensures the kill switch's activation. **4. Potential Impact of IPTG on C. reinhardtii:** **Concern:** Though IPTG is inert in many cellular contexts, we need to consider the potential effects it may have on C. reinhardtii growth or physiology. **Experimental Validation:** To address this, an experiment can be designed where C. reinhardtii cultures are exposed to varying concentrations of IPTG, encompassing our working concentration. By monitoring growth rates, chlorophyll content, and other physiological markers, we can assess any potential negative impacts of IPTG on the health of algae. **5. Operational Mechanism:** **In Laboratory Conditions:** **Activation of Antitoxin Production:** In the presence of IPTG in the culture medium, the Ptrc promoter, controlling the antitoxin gene RelB, becomes highly activated. This leads to a robust expression of the antitoxin, RelB. Due to the strong induction provided by IPTG, the amount of RelB expressed is sufficient to neutralize the effects of the concurrently produced toxin, RelE, ensuring the E. coli cells remain healthy and viable. **Consistent Toxin Production:** The RelE toxin is expressed under the control of the PJ23101 promoter, a moderate-strength constitutive promoter. This guarantees a steady, consistent production of the toxin irrespective of the presence or absence of IPTG. However, in the lab conditions with IPTG present, the high level of RelB antitoxin counteracts this toxin, allowing the bacterial cells to grow normally. **Post-escape Scenario:** **Antitoxin Production Ceases:** In the event the E. coli cells escape into an environment devoid of IPTG, the Ptrc promoter driving RelB expression is not activated. This means the high-level production of RelB antitoxin ceases, dramatically reducing its concentration in the bacterial cell over time. **Continual Toxin Challenge:** The consistent expression of the toxin, RelE, due to the PJ23101 promoter continues irrespective of the external environment. Without sufficient levels of its neutralizing antitoxin, RelB, the harmful effects of RelE start manifesting, leading to bacterial cell death. This serves as an effective safety mechanism, ensuring the genetically modified bacteria cannot survive outside the controlled lab conditions. Auxotrophy Mechanism in C. reinhardtii Using SDH Inhibition:1. Background: The tricarboxylic acid cycle (TCA cycle) is central to cellular metabolism in both prokaryotic and eukaryotic organisms. Disrupting a critical step within this cycle can have profound consequences on an organism's viability. For our kill switch strategy in the eukaryotic algae, we're targeting Succinate Dehydrogenase (SDH) in the TCA cycle, aiming to inhibit its function. This unique approach harnesses the inherent metabolic processes of the cell, making it a feasible and innovative strategy. Our attention is on the inhibition of Succinate Dehydrogenase (SDH), a crucial enzyme in the TCA cycle that oversees the conversion of succinate to fumarate. Oxaloacetate (OAA) has been identified as a competitive inhibitor of SDH. Specifically, OAA binds with a sulfhydryl group of SDH, effectively abolishing enzymatic activity. This bond subsequently evolves into a thiohemiacetal, making the inhibition almost irreversible. With the elevated levels of OAA, the activity of SDH gets inhibited, obstructing the conversion of succinate to fumarate. To naturally increase OAA levels within the cell, we turn our focus to Phosphoenolpyruvate Carboxylase (PEPC). PEPC facilitates the conversion of phosphoenolpyruvate (PEP) to OAA. By amplifying the expression of PEPC, we anticipate a surge in OAA concentrations, culminating in the effective inhibition of SDH. 2. Gene Selection: **Phosphoenolpyruvate Carboxylase (PEPC):** We've chosen the PEPC gene for our approach. PEPC is an enzyme responsible for converting phosphoenolpyruvate (PEP) to oxaloacetate (OAA). By overexpressing this gene, we aim to increase the intracellular concentrations of OAA. Elevated OAA levels will act as a competitive inhibitor for SDH, thereby affecting the TCA cycle and disrupting cellular metabolism. 3. C4-like Pathway in C. reinhardtii: C. reinhardtii, although primarily operating through the C3 photosynthetic pathway, has been found to have the components for a C4-like pathway. PEPC plays a role in this pathway, converting PEP to OAA. By manipulating this pathway and enhancing the activity of PEPC, we can strategically increase OAA levels within the cell. This provides a foundation for our kill switch strategy, allowing us to leverage the native metabolic pathways of the algae for our biosafety objective. 4. Promoter Selection for PEPC Overexpression: **PsaD promoter:** The PsaD promoter from C. reinhardtii is one of the most commonly used promoters for strong, constitutive expression in this alga. It drives the expression of the PsaD gene, which is a part of the photosystem I complex. Given its robust and consistent expression patterns, it seems suitable for our strategy. 5. Determining Optimal Fumarate Concentration: Once the transformation has been validated, the next crucial step would be to identify the optimal fumarate concentration in the growth medium. This ensures the algae’s survival in laboratory conditions while guaranteeing effective containment outside. To determine this, we would culture the transformed algae under varying fumarate concentrations, monitoring growth parameters, especially cell density. The concentration that yields the highest growth rate without any observable stress would be considered optimal. 6. Validation After Successful Transformation: * Gene Expression: Quantitative RT-PCR to measure the mRNA levels of the introduced PEPC gene. * Protein Expression: Western Blot analysis to confirm the expression of the PEPC protein. * Growth Curves: Compare the growth of transformed and non-transformed C. reinhardtii in the presence and absence of external fumarate. 7. Operational Mechanism: **In Laboratory Conditions:** * The transformed algae will be cultured in media supplemented with optimal concentrations of fumarate. * The introduced PEPC gene, driven by the PsaD promoter, will result in elevated OAA levels, inhibiting SDH and causing a disruption in the TCA cycle. * The external fumarate will bypass the blocked step, allowing the algae to grow. **Post-escape Scenario:** * In an environment without supplemented fumarate, the transformed algae will fail to thrive due to the inhibition of SDH by OAA. * This will act as a biological containment mechanism, preventing the algae from growing outside controlled conditions.

# Parts

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### Basic Parts:

| Repository Accession Number | Short Name | Description |
| --- | --- | --- |
| BBa\_K4736000 | pdc (Pyruvate Decarboxylase) | This is the coding sequence for the enzyme pyruvate decarboxylase, which catalyses the conversion of pyruvic acid to acetaldehyde. When used in conjunction with adhB (BBa\_K4736001), it can be used to create ethanol. |
| BBa\_K4736001 | AdhB (alcohol dehydrogenase II) | This is the coding sequence for the enzyme alcohol dehydrogenase II, which catalyzes the conversion of aldehydes to alcohols. In our project, we used this gene in conjunction with pdc (BBa\_K4736000) to synthesize ethanol from acetaldehyde. |
| BBa\_K4736003 | recET (exonuclease VIII) | This is the coding sequence for the enzyme exonuclease VIII, which facilitates homologous recombination of linear DNA between short regions of homology. RecET is analogous to lambda Red recombinase, which also facilitates recombination of short region homology. When paired with regions homologous to a target gene, recET can be used to facilitate a gene knockout. |
| BBa\_K4736004 | ackAF-pta knockout | This part is designed to inactivate E. coli's main acetate production pathway ackA-pta. This part includes the gene for ampicillin resistance, flanked by two short regions homologous to the ackAF and ptaR genes, respectively. When transformed as linear DNA into an E. coli strain expressing recET (BBa\_K4736003), a gene that facilitates the recombination of linear DNA into E. coli's genome, this construct will replace the ackA-pta pathway with the gene for ampicillin resistance, allowing for easy selection of successfully modified bacteria. The goal of inactivating the acetate production pathway is to increase the amount of acetyl CoA available for PHB synthesis. |
| BBa\_K4736005 | phaA | PhaA is the coding sequence for the enzyme acetyl-CoA acetyltransferase. This enzyme catalyzes the conversion of acetyl-CoA into CoA and acetoacetyl-CoA. It is the first step in converting acetyl-CoA into polyhydroxybutyrate (PHB). |
| BBa\_K4736006 | phaB | This gene codes for the enzyme acetoacetyl-CoA reductase. This enzyme catalyzes the reduction of acetoacetyl-CoA to β-hydroxybutyrate, the second of three steps for PHB synthesis from acetyl-CoA. |
| BBa\_K4736007 | phaC | This gene codes for the enzyme polyhydroxybutyrate polymerase. This enzyme catalyzes the polymerization of β-hydroxybutyrate into polyhydroxybutyrate, or PHB, the last of three steps in the production of PHB. |

## Composite Parts

| Repository Accession Number | Short Name | Description |
| --- | --- | --- |
| BBa\_K4736002 | pdc & adhB for ethanol synthesis | This is a composite part for ethanol synthesis in E. coli. It expresses two coding sequences, pdc (BBa\_K4736000) and adhB (BBa\_K4736001), which together facilitate the conversion of pyruvic acid into ethanol. It is controlled by the constitutive promoter BBa\_J23105. This is the first iteration of our expression part, and we plan to test it under the control of different promoters. |
| BBa\_K4736008 | mCherry expression | We designed this part to express mCherry in C. reinhardtii to quickly indicate if our transformation was successful. This part includes the promoter CaMV 35S, mCherry, 3'UTR, and terminator. |
| BBa\_K4736009 | phaA expression | This composite part expresses phaA (BBa\_K4736005) using the promoter CaMV35S (BBa\_J428074) and a eukaryotic terminator (BBa\_J428081). |
| BBa\_K4736010 | phaB expression | This composite part expresses phaB (BBa\_K4736006) using the promoter CaMV35S (BBa\_J428074) and a eukaryotic terminator (BBa\_J428081). |
| BBa\_K4736011 | phaC expression | This composite part expresses phaC (BBa\_K4736007) using the promoter CaMV35S (BBa\_J428074) and a eukaryotic terminator (BBa\_J428081). |

{% endblock %}

{% 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 %}

## Culturing *C. reinhardtii*

From https://2016.igem.org/Team:Linkoping\_Sweden/Protocols#Cultivation\_of\_algae

**Material**

1. 100 ml TAP – medium

2. 1ml Alg suspension

**Procedure**

1. Mix 100 ml TAP – medium with 1 ml alg suspension

2. Incubate the culture on a shaker (or pump if available) (100 - 150 rpm, LED light 14 hours/day, temperature constant 22 °C)

Measure the growth using a spectrometer (750 nm) every day about the same time. Incubate until the growth curve reaches 0.5 (6).

## *E. Coli* Transformations

Transformations (Electroporation or Heat Shock)

**To thaw:**

1. Competent cells (on ice) … Found in -80 freezer

2. SOC medium (@ room temp.) … Found in -80 freezer

3. Turn on shaking incubator

4. Turn on water bath (Heat shock only)

5. Agar plates in incubator

The DNA (@ room temp)

**Electroporation**

1. Combined 40 μL of electrically competent DH5a cells and 1 μL of ligated DNA to an Eppendorf tube.

2. Transferred the contents of the Eppendorf tube to a cuvette and lightly tapped the cuvette on the table to evenly distribute the contents and to get rid of air bubbles.

3. Placed the cuvette into the Bio-Rad MicroPulser and delivered the electric shock.

4. Immediately after, added 900 μL SOC medium to the cuvette and micropipette mixed the solution.

5. Transferred the solution from the cuvette to a shaker tube and placed in the shaker at 37°C at 200 rpm for 1 hour.

6. After shaking for 1 hour, streaked 150 μL of the solution onto an agar plate with the respective antibiotics.

7. Incubated plates at 37°C for at least 24 hours.

**Heat Shock**

1. Thawed One Shot TOP10 chemically competent cells on ice (50 ul).

2. Added 2 μL of DNA sample into competent cells

3. Incubated the cells on ice for 35 minutes.

4. After the ice incubation, placed the samples into a 42° C water bath for 30 seconds.

5. Quickly took them out and immediately added 250µL of SOC medium

6. Place the samples into a 37° C shaking water incubator for 1 hour at 200 rpm.

7. After shaking for 1 hour, streaked 150 μL of the solution onto an agar plate with the respective antibiotics.

8. Incubated plates at 37°C for at least 24 hours.

## **Gel Electrophoresis**

**Preparing, Loading, and Running a 1% Agarose Gel**

**Things to Thaw:**

1. MW ladder (not on ice) … found in freezer

2. Loading Dye (not one ice) … found above scale

**Preparing**

1. Added 1 g of Agarose in 100 mL of 1X TBE in an Erlenmeyer flask

2. Heated in the microwave until fully dissolved (usually about 45 seconds to 1 minute)

a. Solution should be completely clear

b. Do in 30 second intervals

3. Allowed the solution to cool until comfortable to touch

4. Added 10 μL GelRed Nucleic Acid Gel Stain and mixed

5. Inserted casting tray, made sure the rubber on the sides is not overlapping

6. Carefully poured the agarose into the tray and placed the comb to create the wells

7. Allowed the gel to solidify

8. Once solidified, changed the orientation of casting tray where the rubber sides are not in contact with the sides of the system.

9. Poured in 1X TBE into the gel electrophoresis system to the fill line, being sure to submerge the gel, and removed the comb

**Loading**

1.Loaded ~5 μL of the ladder in the first well

2. Prepared your samples to load by adding in 1 μL of 5X Loading dye for every 5 μL of DNA and loaded

**Running**

1. Once the gel had been loaded, slid on the cover making sure the negative electrode is closest to the DNA and the positive electrode is at the bottom of the gel

2. Ran for about 45 minutes to an hour

a. Note: Longer with lower voltage gets you a better gel

## **Gel Extraction**

**Qiagen QIAEX II Gel Extraction Kit**

1. Ran a restriction digest on the targeted DNA part using restriction enzymes and ran an agarose gel overnight

2. Cut the targeted DNA sequence out using a razor blade, made sure to get as much DNA while limiting the amount of agarose extracted

3. Pre-weighed an empty 50 mL falcon tube before adding the gel exicisions.

4. Added the gel extracts to the Eppendorf tubes and weighed again.

5. Calculated the mass of the gel using the difference of the two measurements.

6. Multiplied the mass by a factor of 3 to get the volume of Buffer QG needed.

7. Added the respective amounts of Buffer QG to each of the tubes

8. Vortexed the tubes until all the gel had dissolved

9. Added 1:1 ratio of Gel mass: Isopropanol

10. Once dissolved, added the solution into spin columns and centrifuged for 30 second at 13,000 rpm and carefully removed the supernatant.
Note: One column can only handle .4 grams of agarose

11. Added 375 μL of Buffer QG to each of the spin columns, centrifuged for 1 minute, and discarded the flow through

12. Added 750 μL Buffer PE to each of the spin columns, centrifuged for 1 minute, and discarded the flow through

13. Centrifuged the spin columns empty to remove the residual buffer.

14. Added 50 μL Buffer EB to each of the spin columns and eluted the DNA into two different clean eppendorf tubes.

## **Lab Prep**

**10X TBE**

1. Dissolved 108 g Tris and 55 g Boric acid in 800 mL of diH2O

2. Added 40 mL 0.5 M Na2EDTA (pH 8.0)

3. Brought final volume up to 1 L

**Terrific Broth media**

1. 450 mL of deionized water was added to a 2L flask

2. 24 g of yeast extract , 20 g of tryptone, and 4mL of glycerol were added

3. The solution was stirred until the ingredients dissolved

4. The flask was covered with aluminum foil and autoclaved for 45 minutes

5. The solution was cooled

6. 100 mL of phosphate buffer was added and it was stored in the fridge

**LB media**

1. 10 grams Tryptone

2. 10 grams NaCl

3. 5 grams Yeast Extract

4. Filled up to 1 L with di wat

5. Autoclaved for 45 minutes

**YM media**

1. Dissolved the following in 900 mL of diH2O and bring up to 1 L

a. 0.4 g Yeast extract

b. 10 g Mannitol

c. 0.1 g NaCl

d. 0.2 g MgSO4ï 7H2O

e. 0.38 g K2HPO4

2. Autoclaved for 45 minutes and bring to a pH of 7

## **Ligation**

Purpose: To add a gene into a plasmid

1. Added 6 μL of diH2O to a clean 1.5 mL Eppendorf tube, 1μL of T4 DNA Ligase Buffer, 1μL of plasmid DNA and 1μL of DNA part and mixed.

2. Added 1μL of T4 DNA Ligase.

3. Pipet mixed the tube and incubated at room temperature for 10 minutes.

## **Mini Prep**

**QIAprep Spin Miniprep Kit Protocol**

1. Centrifuged 3 mL of bacterial overnight culture in two separate Eppendorf tubes (1.5 mL in each) at 8,000 rpm for 3 minutes at room temperature.

2. Discarded the supernatant and resuspended pelleted bacterial cells in one tube with 250 μL Buffer P1 and transferred to the other and resuspended until one eppendorf tube contained the pelleted cells resuspended in 250 μL Buffer P1.

3. Added 250 μL of Buffer P2 and inverted 5 times.

4. Added 350 μL of Buffer N3 and immediately mixed by inverting 5 times.

5. Centrifuged for 10 minutes at 13,000 rpm.

6. Micropipette 800 μL of the clear supernatant into a spin column and centrifuged for 60 seconds and discarded the excess liquid.

7. Add 500 μL of PB and centrifuged the spin columns for 60 seconds. Discarded the flow through.

8. Added 750 μL of PE to the spin columns, centrifuged for 60 seconds, and discarded the flow through.

9. Centrifuged the spin columns again for 60 seconds to remove residual wash buffer and discarded the flow through.

10. Transferred the spin columns to a clean eppendorf tube and added 50 μL of EB to the center of the spin column to elute the DNA.

11. Allowed the spin column to stand for one minute and then centrifuged for one minute.

12. Recorded the concentrations for each sample./p>

## **Overnight Cultures**

Purpose: To grow bacteria containing the introduced plasmid

**To Thaw:**

1. Antibiotics (Thaw @ room temp.) … Found in freezer

1. Added about 5-7 mL of LB to a 25 mL Falcon tube along with 5-7 μL of antibiotics (Chloramphenicol for pSB1C3; Kanamycin for pCB302; Ampicillin for DinoIII)

2. Dipped a p10 tip into your selected colony and dropped into the tube

a. If using a glycerol stock, simply scrapped some of the ice with the p10 tip and dropped into the tube

i. NOTE: did NOT let the glycerol stock defrost!

b. If you want to do overnight colony PCR from the same colonies:

i. Used the tip of a pipette or toothpick to pick a colony then swirled it around in 10 μL of DI water

ii. Added 1 μL into PCR tube and another μL into overnight cultures

3. Incubated in the water bath at 37° C at 220 rpm for 16-18 hours

Purpose: To save your cells with the introduced plasmid

Glycerol Stocks

1. Took 1 mL of 50% glycerol and 1 mL of the overnight culture (after incubation) and added to a glycerol stock tube.

2. Labelled with your name, date, and the contents and stored in the -80° C freezer in CLSO 442

## **PCR Protocol**

Purpose: To amplify a sequence of DNA

**To thaw:**

1. Mastermix (on ice) … Found in freezer

2. Primers (on ice) … Found in freezer

**20 μL Reaction**

1. Prepared a PCR concentration cocktail with the following proportions: 7 μL of diH2O, 10 μL PCR Mastermix, 1 μL of the forward primer, and 1 μL of the reverse primer.

2. Added 19 μL of the concentration cocktail into a PCR tube along with 1 μL of the DNA.

2. Placed PCR tube in the thermocycler at the following generic settings:

a. 95° C f or 3:00 minutes

b. 95° C for 1:00 minute

c. 52° C for 1:00 minute \*Annealing temperature varies depending on primer

d. 72° C for 1:00 minute

e. 30X (Go to Step 2)

f. 72° C for 5:00 minutes

Lid Temperature 105° C

**Colony PCR Protocol**

1. Prepared a PCR concentration cocktail with the following proportions: 7 μL of diH2O, 10 μL PCR Mastermix, 1 μL of the forward primer, and 1 μL of the reverse primer.

2. Added 19 μL of the concentration cocktail into a PCR tube.

3. Using a 10 μL micropipette, touched the tip onto the selected colony and swirled around in the PCR tube.

4. Placed PCR tube in the thermocycler at the following generic settings:

a. 95° C for 3:00 minutes

b. 95° C for 1:00 minute

c. 52° C for 1:00 minute \*Annealing temperature varies depending on primer

d. 72° C for 1:00 minute

e. 30X (Go to Step 2)

f. 72° C for 5:00 minutes

Lid Temperature 105° C

## **Restriction Digest/Heat Kill**

Purpose: to cut at restriction sites and remove inserted DNA from plasmid.

**To thaw:**

1. Restriction enzyme (on ice) …. Found in freezer

2. Fast Digest buffer (on ice) … Found in freezer

3. Turn on water bath

30 μL Fast Digest Restriction Digest

1. Prepared a Fast Digest concentration cocktail with the following proportions: 1 μL Restriction Enzyme #1, 1 μL Restriction Enzyme #2, 3 μL of 10X Fast Digest Buffer, and 15 μL of diH2O.

2. Added 20 μL of this cocktail to a clean 1.5 Eppendorf tube and then added 10 μL of DNA

3. Incubated at 37° C for 30 minutes.

**Restriction digest protocol**

Purpose: To digest/linearize large amounts of plasmid for gel extraction

250 μL Fast Digest Restriction Digest

4. Prepared a Fast Digest concentration cocktail with the following proportions: 25 μL Restriction Enzyme #1, 25 μL of 10X Fast Digest Buffer, and 100 μL of diH2O.

5. Added 20 μL of this cocktail to a clean 1.5 Eppendorf tube and then added 100 μL of DNA

6. Incubated at 37° C for 3 hours.

**Heat Kill**

Purpose: To inactivate the restriction enzymes before a ligation is performed

1. Placed digested DNA in water bath at 65° C for 20 minutes

{% endblock %}

{% 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 %}

## Results

---

### *C. reinhardtii* Culture

Our 2023 iGEM project is the first time our lab has attempted to culture *C. reinhardtii*, and we are excited to have successfully cultured this microalgae. We based our protocol on the 2016 Linkoping University iGEM team, and we found that this protocol worked well for our lab.

### Parts Construction

We successfully transformed our genes for PHB expression, ordered from IDT, into *E. coli*. To
begin building our expression part, we successfully transformed CaMV35S (BBa\_J428074),
mCherry (BBa\_J428079), and eukaryotic terminator (BBa\_J42808) into *E*. We verified that our
transformation was successful through colony PCR and extracted the plasmid through mini-prep.

**Figure 1** Left- Gel results simulated in SnapGene. Lanes 1 contains the eukaryotic terminator.
Lanes 2 and 3 contain mCherry. Lanes 4 and 5 contain CaMV35S. Right- Our experimental gel
showing colony PCR results is consistent with our simulated gel.

![Figure 1](static/assets/Results-01.jpg)

With a large amount of our kit plate parts secured, we began a process of restriction digest and
ligation to attach these genes to our IDT-synthesize PHB genes. We designed four expression
parts, one for phaA (BBa\_K4736009), phaB (BBa\_K4736010), phaC (BBa\_K4736011), and our
reporter gene mCherry (BBa\_K4736008).

**Figure 1** mCherry composite part for expression

![Figure 1](static/assets/Results-02.jpg)

**Figure 2** phaA composite part for expression

![Figure 2](static/assets/Results-02.jpg)

**Figure 3** phaB composite part for expression

![Figure 3](static/assets/Results-03.jpg)

**Figure 4** phaC composite part for expression

![Figure 4](static/assets/Results-04.jpg)

However, after transforming our ligated expression part into E. coli and running colony PCR, our
experimental gel does not show the expected results, suggesting that contamination may have
occurred. We will begin the restriction digest and ligation process again, and we expect to have a
successful transformation and have tested for PHB production by the 2023 iGEM Jamboree.

**Figure 5** Gel results simulated in SnapGene. Lanes 2 and 3 contain the phaA expression part.
Lanes 4 and 5 contain the phaB expression part. Lanes 6 and 7 contains the phaC expression
part. Lanes 8 and 9 contain the mCherry expression part.

![Figure 4](static/assets/Results-05.jpg)

{% endblock %}