iGEM 2022 — Team asu

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[ASU iGEM 2022](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27index%27%29%20%7D%7D)

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Notebook

# May/June

![](https://static.igem.wiki/teams/4455/wiki/junenotebook.png)

# October

![](https://static.igem.wiki/teams/4455/wiki/octobernotebook.png)

# July

![](https://static.igem.wiki/teams/4455/wiki/julynotebook.png)

# September

![](https://static.igem.wiki/teams/4455/wiki/septembernotebook.png)

# August

![](https://static.igem.wiki/teams/4455/wiki/augustnotebook.png)

May/June

×

MAY 1-7

- End of recruitment period
 - Finalized initial project idea and approach
 - Lab safety
training with Dr. Bartelle
 - Established preliminary project deadline
 - Compiled
pre-existing basic lab protocols from 2021 (Western Blot,  *C. reinhardtii  *transformation)
- Researched filter prototyping basics**

MAY 8-14

- First official team meeting
 - Researched genes of interest and began uploading sequences to
Benchling
 - Troubleshot in-silico virtual assemblies of genes of interest
 - “Work Sessions”
to make progress on codon-optimization of genes of interest and running Golden Gate assemblies
in-silico
 - Determined goals for human practices team

MAY 15-21

- Began brainstorming water filter ideas related to hydrocyclone separation
 - Purchased lab
supplies
 - Troubleshot construct design
 - Researched 2A peptide sequences for use in  *C.
reinhardtii*

MAY 22-28

- Delegated human practices tasks based on previously discussed goals
 - Finalized and ordered
constructs

MAY 29 - JUNE 4

- Researched MoClo kit for  *C. reinhardtii  *- Re-ordered constructs after further
troubleshooting
 - Purchased more reagents and lab supplies
 - Met with team to discuss
final construct design
 - Round 1 of sketching out filter designs
 - Wrote out basic lab
protocols
 - Collaboration meeting with QGEM
 - Updated project timeline**

JUNE 5-11

- Researched FRET protocol
 - Started discussing collaboration with UBCO iGEM
 - Initial CAD
attempts for filter design
 - Wrote industrial stakeholder profiles to prepare for human practices
interviews
 - Received  *Chlamydomonas Reinhardtii  *MoClo Toolkit and prepared cultures for
glycerol stocks as well as minipreps of parts in the kit needed for our project**

JUNE 12-18

- Received vector to be used in our project and prepared a culture for a glycerol stock as well as a
miniprep of it First attempt at Golden Gate Assembly of plasmid containing genes of interest was
unsuccessful
 - Second attempt at Golden Gate Assembly of plasmid yielded red colonies resulting
from an mScarlet reporter, which were picked and subjected to restriction digestion analysis
 -
Designed variants of potential filter designs
 - Safety forms
 - HP stakeholder meeting with
Dr. Bruce Rittmann
 - Collaboration meeting with SBU iGEM
 - Wrote scientific stakeholder
profiles to prepare for human practices interviews
 - Began planning Phototroph Community with
UBCO
 - Collaboration meeting with iGEM Concordia

JUNE 19-25

Constructs from the second attempt at Golden Gate assembly which were confirmed by restriction
digestion analysis were subjected to an additional one-fragment Golden Gate assembly in which the
mScarlet reporter was replaced with a TaV 2A peptide, yielding the final construct
 - The final
construct, named A2, underwent restriction digestion analysis in order to confirm correct assembly

 - Phototrophs outreach and recruitment
 - Finished safety forms
 - QGEM podcast outreach
and interview requests
 - Initial wiki planning and drafts
 - Filter prototyping and
conceptual design sessions

JUNE 26 - JULY 2

- Our final A2 construct was additionally confirmed as correct by whole-plasmid DNA sequencing
- Tested glass bead transformation with unsequenced plasmid containing antibiotic resistance gene
- Researched assays to confirm successful transformation for final plasmid
 - Wrote article for
SBU journal collaboration
 - CAD designs for filter, continued

July>

October

×

OCTOBER 2-8

- The FLAG blot from the last Western Blot trial was reprobed, confirming that the bands apparent on
it were background and did not result from the production of the tagged protein of interest
 -
Completed 4-day testing cycle to determine arsenic uptake abilities of mutant strains
 - Edited
phototroph handbook and video protocol submissions

OCTOBER 9-12

- Wiki writing
 - Data analysis
 - Model validation
 - Podcast editing

July

×

JULY 3-9

- First attempt at glass bead transformation with sequenced plasmid proved unsuccessful
 -
Podcast interview #1 - Dr. Stephen Brown
 - Began planning modeling strategy
 Defining and
finding samples of parts needed for water filter
 - Met with UBCO to plan Phototroph Community
meetings for the summer
 - More podcast outreach

JULY 10-16

- First attempt at transformation via electroporation with sequenced plasmid proved unsuccessful

 - Researched specific parts needed to maintain algal growth in water filtration system
 -
Podcast Interview #2 - Dr. Arul Varman
 - Submitted IRB application for approval

JULY 17-23

- Resolved contamination issue in  *C. reinhardtii  *agar plates
 - Podcast interview #3 -
Dr. Rosy
 - Received IRB approval
 - Modeling strategy and meeting with Dr. Andrew Marcus

 - Sequencing plasmids
 - Phototroph community outreach and planning
 - Visited Dr.
Weiss at ASU’s AzCATI facility
 - Filter prototyping: researched sedimentation strategy after
rethinking approach**

JULY 24-30

- Continued attempts at transformation of  *C. reinhardtii  *with A2 plasmid via the glass bead
protocol
 - Preparation for first Phototroph Community meeting
 - Transcribed podcasts for
later editing
 - Promotional video preparation
 - Refined modeling approach and researched
existing models of similar systems**

JULY 31 - AUGUST 6

- Planned bioremediation conference with iGEM Patras and iGEM Tec-CEM
 - Ordered new strains of
 *C. reinhardtii  *for further testing and troubleshooting
 - Moved base of operations to Dr.
Redding’s lab
 - Tested transformation and material preparation
 - Continued glass bead
transformation trials of  *C. reinhardtii  *and established transformation log to track
protocol changes and time required for transformant colonies to appear
 - iGEM online
meetup hosted by iGEM Worldshaper-HZ****

August>

September

×

SEPTEMBER 4-10

- Chlamy maintenance while preparing for assays
 - First two attempts of colony PCR as described
by Nouemssi et al. 2020 on successful CC-400 transformants containing A2 were unsuccessful
 -
Prepared for transformations of 2A peptide test plasmids

SEPTEMBER 11-17

- First attempt at colony PCR using Promega Wizard Genomic DNA Preparation Kit followed by PCR was
successful and indicated three positive transformants
 - Finished handbook and video protocol
contributions to phototroph handbook
 - Miniprepped plasmids needed for 2A peptide testing
 -
Began coordinating with Dr. Westerhoff’s lab for running ICP-MS on our samples

SEPTEMBER 18-24

- Completed 15 transformations of 2A peptide test plasmids for FRET testing
 - Prepared cultures
of positive cPCR hits for western blotting
 - A protein lysate of one of the positive cPCR hits
has prepared and a BCA assay was performed
 - A Western Blot using the protein lysate from the
positive cPCR hit was performed yet bands were only apparent for the control, not the tagged proteins
of interest

SEPTEMBER 25 - OCTOBER 1

- Protein lysates were prepared from cultures of all three positive cPCR hits and a BCA assay was
performed
 - A Western Blot using the protein lysate from the positive cPCR hit was performed, and
bands were apparent on the FLAG blot as well as the control blot but not the HA blot
 - Another
Western Blot was performed using the same protein lysate samples with a skipped lane between the wild
type and transformant  *C. reinhardtii  *protein lysate samples to prevent contamination of the
untransformed lane, and additionally, a control was included to confirm the efficacy of the
antibodies used
 - Transformants of “FRET plasmids” appeared on plates**

October>

August

×

AUGUST 7-13

- Recorded promotional video
 - Podcast outreach for final interviews
 - Phototroph
community meetup 2 preparation
 - Continued glass bead transformation trials of  *C. reinhardtii
 *with alterations including testing the Kindle protocol (1990) with SGII media as well as the Kao
and Ng protocol (2017) with polyethylene glycol**

AUGUST 14-20

- Continued glass bead transformation trials of  *C. reinhardtii  *using the Kindle protocol
(1990) with SGII media as well as the Kao and Ng protocol (2017) with polyethylene glycol
 -
Phototroph community meetup 2 preparation
 - Phototroph community meetup 2 (theme:
troubleshooting)
 - Outreach for HP stakeholder meetings**

AUGUST 21-27

- Obtained transformants of CC-400 strain of  *C. reinhardtii  *and A2 plasmid
 - HP
stakeholder meeting 1 & 2
 - Phototroph community virtual conference preparation
 -
Started cultures of transformant strains for further testing
 - Prepared for colony PCR**

AUGUST 28 - SEPTEMBER 3

- Podcast interview #4 - Dr. Sundaram
 - Began working on handbook and video protocol
contributions to phototroph handbook

September>

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Engineering Success

Introduction

The construction of our transgenic vector for transformation into *C. reinhardtii* involved
the use of 2A “self-cleaving” peptide sequences, the vast majority of which have not been characterized for
use in *C. reinhardtii*. Our team decided to test five different 2A peptide sequences to determine
which one had the highest cleaving efficiency and would therefore be best to use in a multicistronic
expression vector.

Design: Initial Design of Multicistronic Vector

The genes we planned to insert into the *C. reinhardtii* nuclear genome were genes encoding
for phytochelatin synthase (PCS), arsenate reductase (ACR2p), and paromomycin resistance (ParoR).

Several strategies were undertaken to increase the nuclear transgene expression of the PCS and
ACR2p genes to be integrated into our construct. Altering factors such as promoters, introns, codon usage, or
terminators has been shown to have the potential to improve nuclear transgene expression in *C.
reinhardtii* [1]. The PCS and ACR2p genes to be used were codon optimized in accordance with
the codon bias for *C. reinhardtii*. This codon optimization step is crucial for high transgene
expression as transcripts with improper codon usage are not only less translated, but also, they are more
likely to be degraded [1]. Additionally, introns were inserted into the codon-optimized PCS and
ACR2p genes in order to further increase transgene expression. This increase in transgene expression results
from enhancer sequences in intron sequences as well as the increase in transcript levels via intron mediated
enhancement [1]. The ParoR gene was a component taken from the Chlamydomonas MoClo Toolkit and thus
was already codon optimized for use in *C. reinhardtii* and contained introns.

To the PCS, ACR2p, and ParoR genes which were optimized for nuclear transgene expression in *C.
reinhardtii*, further elements were added which would aid in cloning and analysis of the transformants.
FLAG and HA tags added to PCS and ACR2p respectively served to enable protein production to be detected via
western blotting. In addition, Golden Gate restriction enzyme sites containing overhangs compatible with the
Chlamydomonas MoClo Toolkit were added to PCS and ACR2p in order to aid plasmid construction. Restriction
enzyme sites were not added to ParoR as it was included in the Chlamydomonas MoClo Toolkit and already
possessed these.

In our project, we not only planned to design a construct containing our genes of interest, but
also, we aimed to expand the number of 2A peptides available for genetically engineering in *C.
reinhardtii* by testing them via Förster resonance energy transfer (FRET). To do this, we designed a
construct containing mCerulean, mVenus, and ParoR from the Chlamydomonas MoClo Toolkit. In this construct,
mCerulean and mVenus would be separated by an experimental 2A peptide not well characterized in *C.
reinhardtii*, and mVenus and ParoR would be separated by FMDV, a 2A peptide demonstrated to be
functional in *C. reinhardtii* and included in the *C. reinhardtii* MoClo Toolkit.

![](https://static.igem.wiki/teams/4455/wiki/screen-shot-2022-12-12-at-2-31-26-pm.png)

*Figure 1. The 2A peptide mechanism produces independent polypeptides under the control of a single promoter.*

The multiple different iterations of plasmids used in our FRET testing all possessed the same
structure aside from the experimental 2A peptide which was varied between each plasmid. In order to avoid
building the same construct multiple times, we created a modular plasmid containing all of the parts which
would remain consistent throughout each plasmid as well as a mScarlet reporter in the place of the
experimental 2A peptide. This mScarlet reporter, a visual selection marker which caused successful *E.
coli* colonies to fluoresce red, not only enabled us to detect the successful assembly of this modular
plasmid, but it also allowed us to easily switch it out with different experimental 2A peptides, as it was
designed to be flanked by Golden Gate restriction enzyme sites. All of the 2A peptides which were used in this
project were designed to be flanked with compatible Golden Gate restriction enzyme sites. A similar modular
design was employed in the plasmid containing our genes of interest to enable us to easily insert a new
experimental 2A peptide into our construct after determining the most effective 2A peptide via FRET testing.

Build: Golden Gate Assembly

Prior to constructing the final constructs for our genes of interest as well as our 2A peptide FRET
testing, the modular constructs containing the mScarlet reporter were constructed for each. To build the
modular construct for the genes of interest, a five-fragment Golden Gate assembly was performed with PCS, the
mScarlet reporter, ACR2p, FMDV, and ParoR. The modular construct for FRET testing was constructed by
assembling the following parts: mCerulean, the mScarlet reporter, mVenus, FMDV, and ParoR.

After the modular constructs for our genes of interest and our 2A peptide FRET testing were
constructed, various 2A peptides were inserted via one-fragment Golden Gate assemblies. The TaV 2A peptide was
inserted into the genes of interest modular construct. The TaV, TME-GD7, DrosC, HuRV, and IFV 2A peptides were
inserted into the FRET testing modular construct respectively.

Test: FRET Assay:

Once *C. reinhardtii* transformants which successfully integrated the plasmids containing
the fluorophore FRET pair as well as experimental 2A peptides were obtained, they were subjected to FRET
analysis in order to determine the cleaving behavior of the 2A peptides contained therein. The successful
transformants for each of the five FRET plasmids as well as wild type C. reinhardtii were screened in 96 well
plates in a plate reader. Autofluorescence of *C. reinhardtii* was obtained using untransformed, wild
type *C. reinhardtii*. The values of autofluorescence were used to correct the readings obtained from
the FRET plasmid transformants. After performing a Z-test and scoring the data accordingly, it was concluded
that at least one representative transformant from each of the 2A peptides tested exhibited a lack of FRET
activity, indicating cleavage activity of the 2A peptide. While this result is promising, further FRET testing
trials are needed in order to determine the precise cleaving efficiencies of each of the 2A peptides under study.

![](https://static.igem.wiki/teams/4455/wiki/screen-shot-2022-12-12-at-2-36-56-pm.png)

*Figure 2. Successful cleavage of proteins with a 2A peptide results in no detectable FRET.*

Learn: Multicistronic Vector 2.0

While this initial FRET assay provided evidence of successful cleavage activity exhibited in all
five of the tested 2A peptides, we plan to conduct further improved FRET experiments which will enable us to
parse the precise cleaving efficiencies of each of the 2A peptides under investigation. With this data, we
hope to not only improve the number and characterization of 2A peptides available for use in *C.
reinhardtii* engineering, but we also hope to improve the construct containing our genes of interest
used in our own project. After obtaining the relative cleaving efficiencies of each of the 2A peptides under
study, we plan to integrate the most effective 2A peptide into our modular construct, thus producing a
construct containing 2A peptides with the highest cleavage efficiencies in *C. reinhardtii*. Higher cleaving efficiency means that this construct will allow for an increased production of our proteins of interest. Thus, we expect it to result in a measurable improvement in *C. reinhardtii’s* arsenic sequestration rate.

References

[1] M. Schroda, “Good News for Nuclear Transgene Expression in Chlamydomonas,” Cells, 2019.

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Experiments

## LB-amp Plates

Introduction:

This protocol is for the creation of LB-ampicillin plates for use in cloning with  *E. coli.*

Materials:

* LB-agar powder
* Double-distilled water (ddH2O)
* Wooden sticks
* Aluminum foil
* Ampicillin
* Culture plates/petri dishes

Procedure:

1. Measure 17-20 g of LB-Agar powder on a scale, and add it to a glass container.
2. Measure 500 mL of ddH2O, and add it to the same container.
3. Stir this mixture gently until homogenous.
4. Place aluminum foil over the lid of the container and label with autoclave tape.
5. Place the container in the autoclave for 20-30 minutes.
6. Remove the container from the autoclave and let the mixture cool until it reaches 55 ˚C.
7. Add ampicillin to the container (100 µg/mL) and stir gently for 30 seconds.
8. Pour a thin layer (about 20 mL) of the LB-ampicillin mixture into culture plates.
9. Let the plates cool for one hour, or until solidified.
10. Store the plates inverted overnight.
11. Label the plates with the correct antibiotic and store in plastic bags at 4 ˚C.

## TAP Media

Introduction:

Tris-Acetate-Phosphate (TAP) is a rich medium used for the suspension of cultures of
*Chlamydomonas reinhardtii*. This protocol makes for 1 liter of 1x TAP media.

Materials:

* 25 mL of 40x Beij
* 25 mL of 40x TAP
* 1 mL of 1000x trace elements

+ EDTA-Na2, 25 mM
+ (NH4)6Mo7O24, 28.5 µM
+ Na2SeO3, 0.1 mM
+ Zn·EDTA, 2.5 mM
+ Mn·EDTA, 6 mM
+ Fe·EDTA, 22 mM
+ Cu·EDTA, 2 mM

* DI water
* Autoclave

Procedure:

1. Combine all reagents and top up to 1 liter of DI water.
2. Autoclave for 30 minutes.
3. Allow to cool before use.

## TAP-paromomycin plates

Introduction:

TAP-paromomycin is a rich medium used for the growth of colonies of *Chlamydomonas
reinhardtii*.

Materials:

* 1x TAP media
* 10 µg/mL Paromomycin
* 15 g/L Agar powder
* Sterile plates

Procedure:

1. Follow the “TAP Media” protocol but add 15 g/L of agar before autoclaving.
2. Add 10 µg/mL of Paromomycin after autocalving 1 liter of TAP and letting cool sufficiently
   for about 30 minutes.
3. Pour warm TAP+Paromomycin in a thin layer enough to cover the whole surface of each plate.
4. Allow media to solidify before keeping in 4C until use.

## Miniprep

Introduction:

Miniprep kit is used to elute DNA from a sample. The handbook dictates that this protocol is
designed for the purification of up to 20 μg of high-copy plasmid DNA from 1–5 ml overnight
cultures of  *Escherichia coli*  in LB medium, but we are using 3mL specifically.

Materials:

* Miniprep kit (QIAprep Spin Miniprep Kit (250) PN: 27106)
* LB amp bacterial plate
* LB amp liquid media
* 37°C incubator
* 37°C shaker incubator
* Microcentrifuge
* Vortex mixer
* 100% ethanol
* Microcentrifuge tubes 2ml sterile
* Microcentrifuge tubes 1.5ml sterile (DNase free)
* Pipettes (1000 uL and 200 μL) and tips

Procedure:

Streak out bacterial plate (Day 1)

1. Streak out bacteria on LB amp plate using the pattern below with three sterile pipette
   tips:

![](https://static.igem.wiki/teams/4455/wiki/streakoutbacteriaplate.png)

Start overnight (O/N) culture (Day 2)

1. Label culture tube with plasmid names.
2. Add 6 ml of LB amp media.
3. Pick a single colony with a pipette tip from a freshly streaked selective plate and drop
   the pipette tip into a correspondingly labeled tube.
4. Incubate for 12–16h/overnight at 37°C on a shaking incubator.

Collect cell pellet (Day 3)

5. Pellet 1.5 ml bacterial culture (not to exceed 15 OD units) by centrifugation at 16,000 x
   g (~13,000 RPM) for 30 seconds. Discard supernatants.

Qiagen Miniprep Kit

***Done once per kit:***

***NOTE:*** Store Plasmid Neutralization Buffer (B3) at 4°C after opening, as
it contains RNase A.

***NOTE:*** Check if precipitate has formed in Lysis Buffer (B2), incubate at
30–37°C, inverting periodically to dissolve.

6. Resuspend pellet in 200 μl Plasmid Resuspension Buffer (B1) (pink). Vortex or pipet to
   ensure cells are completely resuspended. There should be no visible clumps.
7. Lyse cells by adding 200 μl Plasmid Lysis Buffer (B2) (blue/green). Invert the tube
   immediately and gently 5–6 times until the color changes to dark pink and the solution is
   clear and viscous. Do not vortex! Incubate for one minute or less.
8. Neutralize the lysate by adding 400 μl of Plasmid Neutralization Buffer (B3) (yellow).
   Gently invert the tube until color is uniformly yellow and a precipitate forms. Do not
   vortex! Incubate for 2 minutes.
9. Clarify the lysate by spinning for 5 minutes at 16,000 x g.
10. Carefully transfer supernatant to the spin column and centrifuge for 1 minute. Discard
    flow-through.
11. Re-insert column in the collection tube and add 200 μl of Plasmid Wash Buffer 1. Plasmid
    Wash Buffer 1 removes RNA, protein and endotoxin. (Add a 5 minute incubation step before
    centrifugation if the DNA will be used in transfection.) Centrifuge for 1 minute.
12. Add 400 μl of Plasmid Wash Buffer 2 and centrifuge for 1 minute.
13. Transfer column to a clean 1.5 ml microfuge tube. Use care to ensure that the tip of the
    column has not come into contact with the flow-through. If there is any doubt, re-spin the
    column for 1 minute before inserting it into the clean microfuge tube.
14. Add ≥ 30 μl DNA Elution Buffer to the center of the matrix. Wait for 1 minute, then spin
    for 1 minute to elute DNA.

## Golden Gate Assembly

Introduction:

This protocol is intended for use with the Type IIS restriction enzyme SapI. Golden Gate
Assembly is a method of molecular cloning that can easily combine multiple fragments of DNA
inserts. Using Type IIS restriction enzymes, the resulting product is "scarless" and does not
retain the recognition sites, making it essentially irreversible. This protocol was taken from
the DTU Denmark 2019 iGEM team.

Materials:

* T4 DNA Ligase
* 10X T4 DNA ligase buffer
* SapI Enzyme
* Reciever Plasmid
* DNA Fragment with SapI Overhangs
* ddH2O
* PCR Tubes

Procedure:

Reaction Set-Up

1. Add the following reagents to a PCR tube:

* 0.5 uL of T4 DNA ligase
* 2 uL of 10X T4 DNA ligase buffer
* 0.5 uL of SapI enzyme
* 100 ng of receiver plasmid
* Equimolar amounts of inserts
* ddH2O for a total volume of 20 uL

2. Mix the contents of the PCR tube gently by flicking.
3. Place the tube in a thermocycler.

Thermocycler Program

| Step | Temperature | Time |
| --- | --- | --- |

|  |  |  |
| --- | --- | --- |
| 1. Activation of Sapl | 37°C | 5 minutes |
| 2: Activation of T4 ligase | 16°C | 5 minutes |
| Repeat steps 1 & 2 for 25 cycles |  |  |
| 3: Inactivation of SapI | 65°C | 20 minutes |
| 4: Inactivation of T4 ligase | 85°C | 10 minutes |
| 5: Hold | 4°C | Hold |

4. Cleanup any tubes or micropipette tips by disposing in autoclave safe disposable bags and
   autoclave according to autoclave brand guidelines but cycle time must be set for a minimum
   of 30 minutes @ 121C, 15 psi.

## E. coli Transformation

Introduction:

**Escherichia coli** (**E. coli**) is an optimal host for
transformation of DNA into cells. Its growth allows for high expression of desired DNA or
genes of interest. The following protocol is from New England BioLabs® High Efficiency
Transformation Protocol using NEB® 10-beta Competent **E. coli** (High Efficiency)
(C3019H/C3019I) protocol.

Materials:

* NEB 10-beta Competent *E. coli* cells
* SOC media
* LB+ampR plates

Procedure:

1. Thaw a tube of NEB 10-beta Competent  *E. coli* cells on ice for 10 minutes.
2. Add 2 µl containing 1 pg-100 ng of plasmid DNA to the cell mixture (25ml). Carefully flick
   the tube 4-5 times to mix cells and DNA. Do not vortex.
3. Place the mixture on ice for 30 minutes. Do not mix.
4. Heat shock at exactly 42°C for exactly 30 seconds. Do not mix.
5. Place on ice for 5 minutes. Do not mix.
6. Pipette 950 µl of room temperature SOC into the mixture.
7. Place at 37°C for 60 minutes. Shake vigorously (250 rpm) or rotate.
8. Warm selection plates to 37°C.
9. Mix the cells thoroughly by flicking the tube and inverting, then perform several 10-fold
   serial dilutions in NEB 10-beta/Stable Outgrowth Medium.
10. Spread 50-100 µl of each dilution onto a selection plate and incubate overnight at 37°C.
    Alternatively, incubate at 30°C for 24-36 hours or 25°C for 48 hours.

## PCR

Introduction:

This protocol delineates the design of the polymerase reaction and thermal cycles for the
production of many copies of amplified DNA fragments.

Materials:

* Forward/Upstream Primer
* Reverse/Downstream Primer
* Template DNA
* Polymerase Master Mix
* Nuclease Free Water

Procedure:

PCR Reaction Mix

1. Determine size of region of interest

1. Identify gene of interest and record sequence in Benchling including any modifications
   (i.e., terminator sequence)
2. Determine primer size and location including any modifications (i.e., homology regions
   or restriction sites)
3. Add the gene of interest fragment size and primer fragment sizes together to estimate
   total length (or just use benchling tool)

2. Pick a polymerase; Taq polymerase for non-cloning amplification, or high fidelity
   polymerase for cloning

1. Different polymerases have different temperature ranges and times for the reaction.

3. Set up reaction mixture in PCR tube with template DNA, upstream and downstream primers,
   polymerase master mix and nuclease free water

1. Ex. Taq 2x Quick Load PCR Components

| Component | 25 µl reaction | 50 µl reaction | Final Conc. |
| --- | --- | --- | --- |

|  |  |  |  |
| --- | --- | --- | --- |
| 10 µM Forward Primer | 0.5 µl | 1 µl | 0.2 µM (0.05–1 µM) |
| 10 µM Reverse Primer | 0.5 µl | 1 µl | 0.2 µM (0.05–1 µM) |
| Template DNA | variable | variable | < 1,000 ng |
| Taq 2X Master Mix | 12.5 µl | 25 µl | 1X |
| Nuclease-free water | to 25 µl | to 50 µl |  |

PCR Reaction Mix

1. Calculate annealing temperature and extension times and determine thermal cycles

1. Determine GC content and primer concentration to calculate annealing temperature
   (https://tmcalculator.neb.com/#!/main)
2. For extension time, add approximately 30-60 seconds of time per kilobase in amplified
   region of interest
3. For all other times, use higher recommended time
4. Ex. Taq 2x thermal cycler conditions

| Step | Temp | Time |
| --- | --- | --- |

|  |  |  |
| --- | --- | --- |
| Initial Denaturation | 95°C | 3 minutes |
| 30 cycles | 95°C (Denature) | 15-30 seconds 15-60 seconds 1 minutes per kb |
|  | 45-68°C (Annealing) | 15-60 seconds |
|  | 68°C (Extension) | 1 minute per kb |
| Final Extension | 68°C | 5 minutes |
| Hold | 4-10°C | Can stay as long as necessary. Store in 4°C fridge or -20°C for long-term |

Thermal Cycler Conditions

1. Store samples in 4C fridge or freeze in -20C firdge for long-term
2. Add loading dye to sample and run on .7% - 1% agarose gel to confirm amplification

## Glass Bead Transformation

Introduction:

To genetically engineer *C. reinhardtii*, we used the glass bead transformation
method, which has been well-documented as being successful in transforming genes into the
*C. reinhardtii* nuclear genome. Multiple variations of this protocol exist in the
literature, but the protocol below is a mixture of two protocols that gave our team many
transformants.

Procedure:

1. Right before transformation, prepare linearized plasmid DNA via restriction enzyme digest
   using EcoRI to allow for greater chances of transformation in comparison to using circular
   plasmid.
2. Grow C. reinhardtii (strain CC-400, cw-) cell cultures in a baffled Erlenmeyer flask (60
   mL of TAP medium in 125 mL flasks) for 4-6 days prior to transformation or until the cell
   count reaches around 3 x 10^6 cells/mL.
3. Transfer 30-50 mL of cells in TAP into 50 mL falcon tubes and centrifuge them at 3500 x g
   for 10 minutes. The resulting pellet should be easily visible and fully condensed to the
   bottom of the falcon tube.
4. After centrifugation, discard the resulting supernatant in a waste beaker. A dark green
   pellet of C. reinhardtii cells should remain at the bottom of the tube.
5. Re-suspended the pellet with 1/100 volume of TAP gently (if using 50 mL of cells,
   resuspend in 500 microliters TAP = 0.5 milliliters TAP). Avoid excessively agitating the
   cells by slowly pipetting up and down.
6. In pre-autoclaved glass tubes with 300 mg of sterilized glass beads (425-600 micrometers
   in diameter), add the following in order:

1. 2 micrograms of linearized plasmid DNA.
2. The previously resuspended 500 microliters of cells.
3. 5% wt/vol of 20% PEG (polyethylene glycol) (this is ~25 uL if using 50 mL of cells).

7. Add the PEG immediately before vortexing.
8. Vortex the tube at the highest speed for 15 seconds.
9. Transfer the vortexed cells to a 125 mL flask containing 30 mL of TAP medium. Ensure that
   no glass beads are taken up alongside the transformed cells.
10. Place the flask on a shaker at room temperature for 24 hours in the dark to allow the
    cells to recover post-transformation.
11. After the 24 hour cell recovery period, centrifuge the contents of the shaking flask down
    into a pellet.
12. Resuspend the pellet with 50 uL of TAP and spread onto a TAP agar plate with paromomycin
    (or other antibiotic selection marker).
13. Place the plates under direct light. Colonies should appear within 5-14 days
    post-transformation.

## C. reinhardtii Colony PCR

Introduction:

This protocol allows for making many copies of the amplified and transformed *C.
Reinhardtii* colonies.

Materials:

Chelex-100 Solutions Preparation

To prepare 5 mL of 5% Chelex-100 Solution:

* Measure out 5 mL water
* Add 0.25 g Chelex-100

Procedure:

1. Label PCR tubes (individual, 8-strip, or 96-well plate depending on throughput) with
   serial numbers corresponding to the colonies being tested.
2. Dispense 50 µL of the prepared 5% Chelex-100 solution into each of the PCR tubes.
3. Touch each transformant colony with a pipette tip and resuspend it in its respective PCR
   tube.
4. Vigorously vortex the mixture for 10s.
5. Incubate the mixture at 95 °C (in the PCR thermocycler) for 10 min.
6. Cool the plate on ice for 1 minute.
7. Vigorously vortex the mixture for 10s.- Centrifuge the mixture at 3500× g for 5 min at room temperature.
   - Nanodrop supernatant.
   - Perform a PCR reaction (using OneTaq/GoTaq) using 2 µL of the supernatant as the DNA
     template.

## Western Blot

Introduction:

Part of the process of purifying recombinant proteins, Western Blot checks the tagged
constructs to verify expression of our target genes.

Materials:

* 50 mL falcon tubes
* Wash buffer (1 -10 mL)

+ 50mM Tris-HCL pH 7.5
+ 10mM EDTA

* 10x Protease Inhibitors (PI)

+ 100mM EDTA
+ 10mM benzzmidine- HCl
+ 500 ug/ml pepstatin A
+ 200 ug/ml leupeptin
+ 100 uM E64
+ 100 mM e-amino caproic acid, freeze

* XCell SureLock Mini-Cell chamber
* Pre-Cast Nupage 4-12% gel
* 1x MES-TBST Buffer
* CAPS Transfer buffer

+ 200 mL of ethanol
+ 4.43 g CAPS
+ 0.84 g sodium hydroxide pellets
+ Up to 2 L with DI Water

* Owl VEP-2 Electrotransfer Chamber

+ Sandwich + Clip + Sponges

* Immobilin-P transfer membrane (0.2um)
* Whatman paper
* Spatula to crack open pre-cast gel case
* Orbital Shaker
* 5% Milk

+ 100 mL 1XTBST
+ 5 g powdered milk

* 5% Milk and 0.02% Sodium Azide

+ 10mL 5% milk
+ 40 uL Sodium Azide

* Primary Antibodies

+ Dilute into sodium azide solution at appropriate conccentration
+ Can be re-used

* Secondary Antibodies

+ Dilute in 5% milk/TBST solution to appropriate concentration
+ Cannot be re-used

* Film Development Room:

+ Red light
+ Developer machine

* Saran wrap
* Film (Do NOT open box in the light)
* Film Cassette
* WesternBright Sirius HRP Substrate Kit

+ WesternBright SiriusTMLuminol/enhancer solution
+ WesternBright Peroxide Chemiluminescent Detection Reagent

* Scissors

Procedure:

1. Inocluate a Culture of C. reinhardtii

1. Fill a beaker with 40 mL of TAP.
2. User a sterile toothpick to smear a few colonies.
3. Wet the side of the beaker by gently sloshing the TAP, and spin the stick against the
   wet side of the beaker to transfer the colonies into the media.
4. Gently stir and allow to shake at 200 rpm with light until it reaches confluency.

2. Total Cell Lystate

1. Transfer 40 mL of cells into a falcon tube.
2. Spin cells down for 5 minutes at 5000 rpm.
3. Resuspend cells in 1 mL wash buffer.
4. Spin the cells again for 5 minutes in a microcentrifuge tube at 4C.
5. Aspirate the supernatant, removing as much as possible.
6. Resuspend cells to around 10^8 cells/mL with fresh 1x PI. Keep on ice.
7. Freeze in liquid nitrogen overnight.
8. Thaw samples the next day on ice.
9. BCA ASSAY
10. Add 1 volume (=volume of cells in 1x PI) of 2x Laemmli gel sample buffer. Keep on ice
    until all are resuspended.
11. Use pipet tip and then vortex. It will be viscous.
12. Heat at 40C for 30 minutes.
13. Vortex and spin for 5 minutes at full speed in microfuge.
14. Transfer the supernatant to a fresh tube and discard the pellet. Keep on ice until ready
    to load on a gel.

3. Western Blot - Gels

1. Rinse XCell SureLock Mini-Cell chamber with DI water.
2. Rinse pre-cast gel (Nupage 4-12%) with DI water prior to taking it out of the bag.
3. Once pre-cast gel is taken out of the bag, make sure to take the tape off.
4. Place gel into buffer core slot with the front side facing forward.
5. Labeling should be in readable orientation.
6. If not running two gels, place dummy gel or remove slot for second gel.
7. Lock buffer core using gel tension wedge.
8. Fill buffer core with 1X MES-SDS buffer.

1. Wait for ~30 seconds to make sure that gel box is locked properly and buffer is not
   leaking.
2. Continue to fill lower chamber until it is half full.

9. Take out comb from pre-cast gel.
10. Wash out wells using p200 (~100 ul)
11. Load ladder using gel-loading 200 uL pipette tips

1. Load 7-15 ul of sample using gel-loading 200 uL pipette tips
2. Do not leave empy wells next to samples: Fill with Laemmli
3. Only load 7ul of ladder and Laemmli, can load more of sample based on concentration of
   protein (~7 ug)
4. Be careful with the final dispensing bubble

12. Run gel at 120 V \*buffer should fill up the core and cover the electrodes outside of the
    core

1. When starting the electrophoresis, ensure that there are no bubbles
2. Make sure the gel actually starts running

13. Stop the gel once the samples have moved sufficiently down gel (~45 mins. Less if
    smaller proteins)
14. While Gel is Running:
15. Make 2 L of transfer buffer

1. 200 mL ethanol
2. 4.43 g CAPS
3. 0.84 g sodium hydroxide pellets
4. DI water up to 2L
5. Mix using a stir bar

16. After Gel is done running: Rinse Owl VEP-2 and sandwich with DI water
17. Fill Owl VEP-2 with transfer buffer (CAPS)
18. Cut Immobilin-P transfer membrane to appropriate size
19. Cut Whatman paper to appropriate size
20. Fill a bin with transfer buffer to soak both sides of the sandwich
21. Fill a bin with ethanol (large petri dish)
22. When electrophoresis is done, unlock buffer core
23. Remove pre-cast gel from buffer core
24. Rinse pre-cast gel with DI water
25. Use spatula to crack open pre-cast gel case
26. Cut off excess gel (bottom of gel, sides, and top)
27. Set Up Sandwich as follows:

1. 1. Lay down Sandwich with BLACK on bottom
2. 2. Sponge
3. 3. whatman paper

* (1) Roll out whatman paper with serological pipet so that there are no bubbles

4. Place gel (already cut) so that PROTEINS FACE RED.
5. Place transfer membrane on gel, facing proteins.

* (1) Activate transfer membrane prior to using by soaking it in ethanol for ~10
  seconds.
* (2) Use non-serrated forceps to handle transfer membrane
* (3) Make sure it is evenly coated and there are no bubbles (roll out0.

6. Equilibrate transfer membrane with the transfer buffer in the Owl VEP-2
7. Whatman paper

* (1) Roll out whatman paper with serological pipet so that there are no bubbles
* (2) Add some buffer to wet paper

8. Sponge
9. RED on top

28. Lock Sandwich with plastic clip
29. Place sandwich in Owl VEP-2

1. Rubber clip should be facing the top
2. Black side of the sandwich should be facing forward when the electrodes are on the
   right (black side of the sandwich should align with the black elecrtodue, red should
   face red electrode).
3. If possible, place sandwich as close to positive electrode.

30. Top off Owl VEP-2 with transfer buffer
31. Run the electrotransfer at 45 V for 3 hours at 4 C.
32. Remove the sandwich and membrane from sandwich
33. Equilibrate the membrane by placing it in dish with 1X TBST on orbital shaker at 40 rpm
    for ~5 min.
34. Make 5% milk.

1. 100 mL TBST
2. 5 g milk

35. Western Blot - Blocking and Tagging

1. Transport samples to film developer room, and bring the following:

1. waste container
2. saran wrap
3. film
4. cassette
5. 1.5 mL or 5 mL microcentrifuge tubes
6. WesternBright Sirius HRP Substrate Kit

* (1) WesternBright SiriusTMLuminol/enhancer solution
* (2) WesternBright Peroxide Chemiluminescent Detection Reagent

7. p1000
8. scissors
9. timer
10. 1X TBST
11. Marker

2. Turn on developer (should take ~15 minutes to be ready to use)

1. Wait until red "ready" light illuminates
2. When ready to use, place used film in developer to wet rollers

3. Change out 1X TBST and let sit for 5 minutes (second wash)
4. Change out 1x TBST and let sit for 5 minutes (third wash)
5. Prepare enough HRP substrate (1:1) to cover samples.
6. Place membrane on saran wrap and cover samples with HRP substrate for 1 minute.
7. Place membrane on cassette (protein side up).
8. IN THE DARK:

1. cut film in half
2. Cut one corner off of film piece: place this corner in the corner of the cassette
3. Place film in cassette
4. do not shift film once placed, place directlyh on top
5. close cassette for appropriate time according to antibody used
6. place in developer
7. Wait until film is entirely done going through developer
8. TURN LIGHTS BACK ON

9. Mark film

1. Membrane edges
2. Relevant ladder bands
3. Date

36. Stripping/Re-Blocking/Re-Tagging

1. Rinse Membrane 3x in TBST for approx 5 mins each rinse
2. Cover membrane in small amount of antibody stripping solution
3. Place on shaker @ 40 rpm for 5 mins
4. Pour off stripping solution
5. Re-rinse membrane 3 times in TBST for approx 5 mins each rinse
6. Re-block exposing to 5% milk in TBST solution for 1hr or overnight
7. Repeat blocking/tagging section of protocol

## ICP-MS

Introduction:

Inductively coupled plasma mass spectrometry allows for the analysis and quantification of
elements, and in this case arsenic, at trace levels in biological samples.

Materials:

* 7.5% sodium bicarbonate solution

+ 3 g sodium bicardonate
+ 37 mL DI water
+ Filtered to sterlize resulting solution

* 1000x Arsenic stock solution

+ 4.16 g sodium arsenate dibasic heptahydrate
+ 900 mL DI water
+ Mixed to produce resulting solution
+ To be made in chemical fume hood

- Cultures of (3) mutant strains (8622.1.1, 8622.1.2, 8622.3.10) and control CC-400 control strain
- Orbital shaker
- Hemocytometer
- 50 mL falcon tubes
- 15 mL falcon tubes
- TP media
- 125 mL baffled flasks

Procedure:

Sample Preparation and Day 0 collection

1. Gather cultures of three mutant strains (8622.1.1, 8622.1.2, 8622.3.10) and CC-400 control
   strain.
2. Count cells in each culture using a hemocytometer.
3. Normalize each culture to have a standard cell density of 106 cells/mL.
4. Add 50 mL of culture to 4 50-mL falcon tubes each (label the corresponding strain on each
   tube).
5. Spin down cultures at 7100 rpm for 5 minutes at room temperature (21˚C).
6. Discard the supernatant and resuspend the pelleted cells in 1 mL of TP media.
7. Assemble the 12 cultures in 125 mL baffled flasks as follows (there should be 4 samples at
   each arsenic concentration, one for each strain tested):

| Sample concentration (ppb Arsenic) | Culture added (mL) | TP added (mL) | Bicarbonate added (mL) | 1000 x Arsenic stock solution added (uL) | Total volume (mL) |
| --- | --- | --- | --- | --- | --- |

|  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- |
| 50 ppb | 1 mL | 43.2 mL | 0.8 mL | 2 uL | 45 mL |
| 250 ppb | 1 mL | 43.2 mL | 0.8 mL | 10 uL | 45 mL |
| 500 ppb | 1 mL | 43.2 mL | 0.8 mL | 20 uL | 45 mL |

8. The cultures should be labeled as follows:

|  |  |  |
| --- | --- | --- |
| CC-400 (50 ppb) | CC-400 (250 ppb) | CC-400 (500 ppb) |
| 8622.1.1 (50 ppb) | 8622.1.1 (250 ppb) | 8622.1.1 (500 ppb) |
| 8622.1.2 (50 ppb) | 8622.1.2 (250 ppb) | 8622.1.2 (500 ppb) |
| 8622.3.10 (50 ppb) | 8622.3.10 (250 ppb) | 8622.3.10 (500 ppb) |

9. Immediately aliquot 10 mL of solution from each of the samples into 15 mL falcon tubes to
   obtain arsenic uptake levels at t=0.
10. Record the time at which these samples were removed from culture.
11. Count cells in each of these samples using a hemocytometer and record cell densities.
12. Spin down the 10 mL samples in a centrifuge at 7100 rpm for 5 minutes at room temperature.
13. Decant the supernatant into a separate, clearly labeled falcon tube and store safely at room
    temperature.

Day 1 Collection

1. 24 hours after collecting t=0 samples, aliquot 10 mL of solution from each of the samples into
   15 mL falcon tubes to obtain arsenic uptake levels at t=24 hr.
2. Record the time at which these samples were removed from culture.
3. Count cells in each of these samples using a hemocytometer and record cell densities.
4. Spin down the 10 mL samples in a centrifuge at 7100 rpm for 5 minutes at room temperature.
5. Decant the supernatant into a separate, clearly labeled falcon tube and store safely at room
   temperature.
6. Repeat these steps for day 2 and 3 collection.

Safety and Decontamination

1. Arsenic stock solution creation will be done in the Bartelle lab chemical fume hood.
2. All flasks will be thoroughly cleaned and sterilized to remove any residual arsenic
   contamination.
3. All falcon tubes and other disposables that came in contact with arsenic will be disposed of
   in a clearly labeled bin for arsenic waste.

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Results

Below are the results we obtained during the cloning, transformation, and assays performed in the wet lab
to engineer *C. reinhardtii* to uptake arsenic from contaminated water sources.

Golden Gate Cloning and Transformation in *E. coli*

Because we planned to test a number of 2A peptides during this project, we designed our base plasmid to
contain a sequence that we could easily switch out with different 2A peptides. We decided to use the
fluorescent protein mScarlet to act as our “spacer” sequence so it could dually act as a visible selection
marker to confirm that our plasmid had been transformed into *E. coli*. An *E. coli* promoter,
terminator, and ribosome binding site were added to the mScarlet sequence to ensure bacterial expression of
the fluorescent protein. A 7-fragment golden gate assembly was performed to construct the plasmid using the
PICH47742 backbone and was then transformed into NEB 10-beta competent cells. Successful transformants were
expected to appear red after a 24-hour incubation period.

![sequence](https://static.igem.wiki/teams/4455/wiki/r1.png)

*Figure 1. A schematic diagram of the construct containing the mScarlet spacer sequence.*

![GGGG](https://static.igem.wiki/teams/4455/wiki/r2.png)

*Figure 2. An agar plate of *E. coli* transformants containing the mScarlet spacer plasmid
described above. Positive colonies are seen in pink/red.*

Upon successful transformation of the spacer sequence, we then switched out the spacer sequence for the TaV
2A peptide. Because the resulting plasmid no longer contained the mScarlet spacer sequence, transformants
were expected to appear white. This construct was named A2 and was subsequently prepared for transformation
into *C. reinhardtii.*

![UTR](https://static.igem.wiki/teams/4455/wiki/r3.png)

*Figure 3. A schematic diagram of the A2 construct.*

![New](https://static.igem.wiki/teams/4455/wiki/r4.png)

*Figure 4. An agar plate of *E. coli* transformants containing the A2 plasmid described above.
Positive colonies are seen in white.*

Glass Bead Transformation in *C. reinhardtii*

After successfully constructing our plasmid of interest in *E. coli*, we used the glass bead
transformation protocol detailed by Pei-Hsun Kao and I-Son Ng (2017) to transform our plasmid into *C.
reinhardtii* [1]. The glass bead transformation method was chosen because of its previously cited
success in nuclear transformation, and also because our team did not have access to the proper resources to
be able to follow electroporation protocols for *C. reinhardtii* cells. To improve transformation
efficiency, polyethylene glycol (PEG) was added to the glass bead tube before vortexing, and linearized DNA
was used in the transformation. A detailed version of our transformation protocol is on our Experiments
page.

After roughly 2 weeks, transformants were visible on the selective TAP + paromomycin plates.

![hand](https://static.igem.wiki/teams/4455/wiki/r5.png)

*Figure 5. Transformants visible two weeks after following Kao and Ng protocol (with the addition of
PEG) to transform *C. reinhardtii* CC-400 cells.*

View our transformation troubleshooting process on our Contribution [page](https://2022.igem.wiki/asu/contribution).

Colony PCR

After obtaining colonies of CC-400 *C. reinhardtii* cells transformed with the A2 plasmid, we
performed a colony PCR to confirm the presence of our plasmid in the cells. As a control, we used primers
that localized on the phosphoglycerate kinase (PGK) gene that is native to all *C. reinhardtii* cells.
Primers for the colony PCR were designed as outlined in Nouemssi et al. (2020) [2].

Due to the manner in which gene integration occurs in the *C. reinhardtii* nuclear genome, transgene
expression is often hard to predict. As a result, many of the colonies we screened did not show positive
results. We did, however, obtain 3 strains of mutant *C. reinhardtii* that contained our transgenic
plasmid: 8622.1.1, 8622.1.2, and 8622.3.10.

![Part 1](https://static.igem.wiki/teams/4455/wiki/r6.png)

*Figure 6. Round 1 of colony PCR demonstrates that strains 8622.1.1 and 8622.1.2 contain the plasmid
introduced via transformation.*

![Part 2](https://static.igem.wiki/teams/4455/wiki/r7.png)

*Figure 7. Round 2 of colony PCR demonstrates that strain 8622.3.10 contains the plasmid introduced via
transformation.*

View the *C. reinhardtii* colony PCR protocol on our Experiments [page](https://2022.igem.wiki/asu/experiments).

Western Blot

When designing our constructs *in silico*, we decided to tag our two main genes of interest (PCS, ACR2p) to be able to detect them via a western blot. PCS was tagged with a FLAG tag, and ACR2p was tagged with an HA tag. After confirming the presence of our plasmid in three algae transformants (T1, T2, T3) via colony PCR, we performed a western blot on those three transformants to confirm that the proteins corresponding to our genes of interest were being produced in the cell.

Extraction of protein lysates from *C. reinhardtii* cells was performed as outlined in Yildirim (2022) [3]. Western blots were performed a total of three times by our wet lab team with the assistance of Tom Hartley McDermott, Santiago Ochoa, and Bruno La Rosa. The results of our western blot analysis are as follows:

![Part 3](https://static.igem.wiki/teams/4455/wiki/r8.png)

*Figure 8. Western blot attempt 1, 4 minute exposure. FLAG (gene: PCS), HA (gene: ACR2p), β-actin (control), H3 (control) blot results shown.*

The prepared protein lysates of wild type *C. reinhardtii* and our T1 transformant were prepared for western blotting and loaded into the gel in triplicate, enabling the membrane to be tested for the presence of PCS, ACR2p, and control proteins without stripping and re-tagging. The western blot indicated the presence of bands for the H3 control, indicating that the protein extraction and western blotting procedure was successful. It was hypothesized that the absence of bands indicating expression of PCS and ACR2p in the western blot indicated that the T1 transformant, while containing the genes of interest, did not express the proteins of interest.

![Part 4](https://static.igem.wiki/teams/4455/wiki/r9.png)

*Figure 9. Western blot attempt 2, 10 minute exposure. Both FLAG (gene: PCS) and HA (gene: ACR2p) blot results shown.*

The second attempt at the western blot was performed with all three of the *C. reinhardtii* transformants which were confirmed by colony PCR. A primary FLAG antibody which was not HRP-linked was used in this trial, as opposed to the HRP-linked FLAG antibody used in the initial western blot. The membrane tagged with FLAG exhibited bands in each of the transformant lanes. It was hypothesized that the bands in the transformant lanes were due to background activity. However, the lack of a similar band pattern in the wild type lane suggested that these bands were indicative of the production of proteins of interest. Additionally, the three bands apparent on the membrane approximately corresponded to the three different molecular weights resulting from the possible cleaving activity of the 2A peptides in our tricistronic system. Still, a conclusion on the latter hypothesis was unable to be reached unless a wild type lane which was completely free of this banding pattern was obtained.

![Part 5](https://static.igem.wiki/teams/4455/wiki/r10.png)

*Figure 10. Western blot attempt 3, 30 second exposure (FLAG) and 10 minute exposure (HA). FLAG (gene: PCS) and HA (gene: ACR2p) blot results shown.*

In order to confirm whether the banding pattern obtained from the FLAG membrane on the second round of western blots was due to background or indicative of protein expression, an improved western blot using the same protein lysates was performed. In this blot, an empty lane was maintained between the *C. reinhardtii* wild type and transformant lanes. Separation of the wild type and transformant lanes served to prevent possible spillover of sample from the transformant lanes, and this would allow the determination of whether the faint banding pattern observed in the previous trial of western blotting could be attributed to spillover from the transformant lanes. In addition, control samples tagged with FLAG and HA were included on each membrane respectively in order to confirm the functionality of the antibodies used. While the HA membrane was clear as before, the FLAG membrane did not exhibit the same banding pattern it had before and was entirely blank.

![Part 5](https://static.igem.wiki/teams/4455/wiki/screen-shot-2022-10-13-at-7-40-49-pm.png)

*Figure 11. Reprobing of western blot attempt 3 FLAG membrane. FLAG (gene: PCS) blot results shown.*

The FLAG membrane of the western blot was reprobed in order to determine whether a similar banding pattern as that observed previously could be obtained. Reprobing of the FLAG membrane not only yielded the same banding pattern observed in the second western blot attempt, but it also indicated the presence of the same banding pattern in the wild type lanes. This result suggests that the banding pattern obtained in the transformant lanes for the second FLAG western blot was indeed due to background as the wild type lane possesses the same banding pattern. Further western blots should be performed in the future in order to parse out definitively the expression of the proteins of interest in the *C. reinhardtii* transformants.

Arsenic Uptake Experimentation

After results from the Western blot were received, the three transgenic strains (T1, T2, T3) were subjected to arsenic experimentation to determine if our algae was capable of decreasing the arsenic levels in its environment. Experiments were conducted using 50 ppb, 250 ppb, and 500 ppb arsenic, and samples of culture supernatants were taken over the course of 72 hours. Results from ICP-MS testing preliminarily show that our T1 and T2 strains were able to lower arsenic concentrations over the course of 24 hours.

![Part 1](https://static.igem.wiki/teams/4455/wiki/screen-shot-2022-12-12-at-2-41-00-pm.png)

*Figure 12. Comparison of T=0 and T=1 supernatant arsenic concentrations of transgenic strains T1, T2, and T3 as compared to wildtype in 500 ppb arsenic, normalized to cell density. Strains T1 and T2 show preliminary positive results.*

Testing 2A Peptides with FRET

2A peptides are an important tool in synthetic biology which enables the independent expression of multiple proteins in the same transcriptional unit. During translation, ribosomes which encounter 2A peptides will undergo a "ribosomal skipping event," in which a peptide bond fails to be made. This causes a "cleavage" event to take place between the two polyproteins between the 2A peptide sequence, creating two independent polyproteins [4].

While 2A peptides have been widely used in synthetic biology in other chassis, there currently exists a limited number of 2A peptides which have been well characterized and demonstrated to function in *C. reinhardtii* [5]. This relative lack of 2A peptides is highlighted by the inclusion of only one 2A peptide, the FMDV 2A peptide, in the Chlamydomonas MoClo Toolkit. The dearth of 2A peptides currently available for use in engineering of *C. reinhardtii* limits the usage of polycistronic gene expression systems in this chassis. This is disadvantageous as it thus requires that transgenes integrated into *C. reinhardtii* possess separate promoters and terminators, enlarging the size of constructs considerably [6]. In addition, the usage of the same promoters and terminators can cause homologous recombination and gene loss, while the usage of different promoters and terminators can result in differential expression of key genes [6].

In an effort to expand the number of 2A peptides available for use in the engineering of *C. reinhardtii*, we devised an experiment to test the cleaving activity of five novel 2A peptides in *C. reinhardtii* using Förster resonance energy transfer (FRET). In order to perform this experiment, we created five different constructs each containing mCerulean, mVenus, and a paromomycin resistance gene. In each construct, a different experimental 2A peptide was inserted between mCerulean and mVenus, and the well-characterized FMDV 2A peptide included in the Chlamydomonas MoClo Toolkit was placed between the mVenus and paromomycin resistance genes.

![Part 5](https://static.igem.wiki/teams/4455/wiki/screen-shot-2022-10-13-at-7-40-55-pm.png)

*Figure 13. A schematic diagram of the 2A peptide testing construct.*

The mCerulean-mVenus FRET pair allowed the cleavage efficiency of the experimental 2A peptides included in each of the five constructs. If FRET occurs, mCerulean, the donor, will undergo a radiationless transfer of energy to mVenus, the acceptor, when excited [7]. However, the efficiency of this transfer is highly sensitive to the distance between the two fluorophores, making FRET a valuable tool for examining the cleavage efficiency of 2A peptides. If a 2A peptide fails to cleave during translation, the mCerulean and mVenus proteins will be fused, leading to FRET occurrence as a result of their close proximity. If a 2A peptide successfully cleaves during translation, independent mCerulean and mVenus proteins will be produced, and due to the spacing between the fluorophores, FRET will not occur.

In order to perform this experiment, we transformed *C. reinhardtii* independently with the five constructs containing the experimental 2A peptides and performed FRET analysis on our successful transformants. We performed this analysis by screening the successful transformants for mCerulean, mVenus, and FRET activity respectively in 96 well plates using a plate reader. Given that whole cells were used in the analysis, the autofluorescence of untransformed *C. reinhardtii* was accounted for in our analysis. After performing a Z-test on the collected data and devising a metric with which to score the data and distinguish autofluorescence, it was found that at least one representative transformant from each of the 2A peptides tested exhibited a lack of FRET activity, indicating cleavage activity of the 2A peptide. While this experiment does suggest that the five novel 2A peptides are functional in *C. reinhardtii* further testing is necessary to parse the relative cleaving efficiencies of each of these 2A peptides in *C. reinhardtii*.

Conclusions

The outcomes of our assays suggest that though our transgenes were successfully transformed into three of
our *C. reinhardtii* transformants, protein expression may not have occurred. We are currently
performing arsenic uptake experiments on the three mutant strains to further quantify their ability to
sequester arsenic as compared to the unengineered CC-400 strain in case errors were made that prevented us
from seeing expected results on the western blot.

References

[1] P.-H. Kao and I.-S. Ng, “CRISPRI mediated phosphoenolpyruvate carboxylase regulation to enhance the
production of lipid in *Chlamydomonas reinhardtii,* ” Bioresource Technology, vol. 245, pp. 1527–1537,
2017.

[2] S. B. Nouemssi, M. Ghribi, R. Beauchemin, F. Meddeb-Mouelhi, H. Germain, and I. Desgagné-Penix, “Rapid
and efficient colony-PCR for high throughput screening of genetically transformed *Chlamydomonas
reinhardtii*,” Life, vol. 10, no. 9, p. 186, 2020.

[3] A. YILDIRIM, “Fine-tuning of protein extraction from wall-deficient *Chlamydomonas reinhardtii*
using liquid nitrogen and sonication-assisted cell disruption,” Marine Science and Technology Bulletin,
2022.

[4] A. Szymczak, “Development of 2A peptide-based strategies in the design of multicistronic vectors,” *Ashley Publications*, 2005.

[5] T. Plucinak, “Making *Chlamydomonas Reinhardtii* a Better Model Organism: Tackling the Inefficiency of Nuclear Transgene Expression and Improving Methods for the Generation and Characterization of Insertional Mutant Libraries,” *University of Nebraska*, 2013.

[6] T. Souza-Moreira, “Screening of 2A Peptides for Polycistronic Gene Expression in Yeast,” *Fems Yeast Research*, 2018.

[7] “FRET Microscopy with Spectral Imaging,” *Zeiss*, n.d.

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