iGEM 2025 — Team rptu-kaiserslautern

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# Choose Week

Week 1

◀▶

## Week 1

### (KW 45, 04.11.24 – 10.11.24)

Existing Lv. 0 parts diluted to 40fmol (pAR, 3HA-Tag, cCA, ARS2, GLE1,
RPL23) to perform our first Modular Cloning (MoClo). MoClo of our
Contructs cetFv (Cetuximab single cain Fragment variable) and humcetFv
(Humanized Cetuximab single cain Fragment variable) and transformation
into *Escherichia coli (E. coli)* and streak out onto LB-plates for
blue-white selection. Picked colonies for overnight inoculation in LB
media. Miniprep of the cultures for isolation of the MoClo plasmid.
Measuring of the DNA concentration using the Nanodrop. Test digest of
the isolated plasmid with restriction enzyme Bsa1 and gel
electrophoresis of the digested plasmid for visualization. Due to bad
results in the sanger sequencing, repeat of the Lv. 0 MoClo and
following experiments.

![](https://static.igem.wiki/teams/5758/notebook/figure1.webp)

**Figure 1:** Gel electrophoresis of the isolated plasmid DNA of
the Lv. 0 MoClo of cetFv and humcetFv.

## Week 2

### (KW 46, 11.11.24 – 17.11.24)

Analysis of the Lv. 0 MoClo. Gel electrophoresis and sanger sequencing
show the right sequence of nucleotides in the plasmid DNA of
respectively three cetFv transformants and two humcetFv transformants.

![](https://static.igem.wiki/teams/5758/notebook/figure2.webp)

**Figure 2:** Test digest Gel electrophoresis of the isolated
plasmid DNA of the Lv. 0 MoClo of cetFv and humcetFv.

## Week 3

### (KW 47, 18.11.24 – 24.11.24)

LB inoculation of *E. coli* from DMSO stock for the DNA isolation
of
missing Lv. 0 part 514 (SP20). Miniprep of the culture for the isolation
of the part SP20. Preparation for the Lv. 2 MoClo. Design of two
cytosolic constructs with the cetFv and humcetFv parts with no secretion
signal and six constructs with a secretion signal, respectively cetFv
and humcetFv with the secretion signals Carbonic Anhydrase (cCA),
Arylsulfatase (ARS2) and Gametolytic Enzyme 1 (GLE1) for the secretion
of the single chain Fragment variables (scFv). Transformation of the Lv.
2 constructs into *E. coli* for red-with selection. Miniprep
Plasmid DNA
isolation of the transformants and test digest of several colonies.

![](https://static.igem.wiki/teams/5758/notebook/figure3.webp)

**Figure 3:** Test digest and gel electrophoresis of the isolated
plasmid DNA of the Lv. 2 MoClo of cetFv and humcetFv.
Analysis of cytosolic and cCA secretion signal plasmids of the cetFv
and humcetFv MoClo.

![](https://static.igem.wiki/teams/5758/notebook/figure4.webp)

**Figure 4:** Test digest and gel electrophoresis of the isolated
plasmid DNA of the Lv. 2 MoClo of cetFv and humcetFv.
Analysis of ARS and GLE secretion signal plasmids of the cetFv and
humcetFv MoClo

![](https://static.igem.wiki/teams/5758/notebook/figure5.webp)

**Figure 5:** Test digest and gel electrophoresis of the isolated
plasmid DNA of the Lv. 2 MoClo of cetFv and humcetFv.
Analysis of GLE secretion signal plasmids of the humcetFv
MoClo.

## Week 4

### (KW 48, 25.11.24 – 01.12.24)

Sanger sequencing of the cytosolic and secretory constructs. Due to bad
results of the sequencing, repetition of the Lv. 2 MoClo and
transformation into *E. coli*. Miniprep plasmid DNA isolation from
the
transformed *E. coli* cultures. Gel electrophoresis and sequencing
of the
plasmid DNA. Inoculation of *Chlamydomonas reinhardtii* strain
UVM4-Nit in
flasks for transformation next week.

![](https://static.igem.wiki/teams/5758/notebook/figure6.webp)

**Figure 6:** Test digest and gel electrophoresis of the isolated
plasmid DNA of the Lv. 2 MoClo of cetFv and humcetFv.
Analysis of all L2 plasmids of the cetFv and humcetFv
MoClo.

## Week 5

### (KW 48, 02.11.24 – 08.12.24)

Preparation for the first *Chlamydomonas reinhardtii*
transformation via
glass beads. Autoclaving of the glass beads and pouring of the TAP
culture plates. Linearization of the Lv. 2 MoClo plasmids and subsequent
transformation into *C. reinhardtii*. Preparation for a new Lv. 0
MoClo,
design of the same constructs with an added octa His-Tag behind the
HA-Tag. Creation of a HA-Tag version with added mTFP as florescent
marker. MoClo of the Lv. 0 and transformation into *E. coli*.
Miniprep of
the cultures and gel electrophoresis with sanger sequencing for
conformation. Lv. 2 MoClo and transformation into *E. coli*.
Miniprep and
sanger sequencing. Inoculation of *C. reinhardtii* in flasks for a
new
transformation.

![](https://static.igem.wiki/teams/5758/notebook/figure7.webp)

**Figure 7:** Gel electrophoresis of the isolated plasmid DNA of
the Lv. 2 MoClo of cetFv and humcetFv. Analysis of all L2
plasmids of the cetFv and humcetFv MoClo

## Week 6

### (KW 49, 09.12.24 – 15.12.24)

Transformation of the linearized His-Tag and mTFP Lv. 2 MoClo into *C.
reinhardtii* via glass bead transformation. Team internal
organization
events on basic laboratory practice like “how to pour TAP-plates, how to
read sanger sequcencing results with A plasmid Editor (ApE)”.
Inoculation of the HA-Taged *C. reinhardtii* transformants in
flasks.
Pouring of SDS-gels for SDS-PAGE.
 Harvest of the cytosolic *C.
reinhardtii* cultures with no secretion signal from flasks for
screening
via Western Blot. SDS-PAGE and Western Blot of the cytosolic HA-Taged
*C.
reinhardtii* culture. SDS-PAGE failed therefore new inoculation of
*C.
reinhardtii* culture.

## Week 7

### (16.12.24 – 22.12.24)

Pouring of SDS-gels for SDS-PAGE. Harvest of the cytosolic *C.
reinhardtii* cultures (cetFv and humcetFv) with no secretion signal
from
flasks for screening via Western Blot. SDS-PAGE and Western Blot of the
cytosolic HA-Taged *C. reinhardtii* culture. Inoculation of the
mTFP-HA-Tagged *Chlamydomonas* in flasks for florescence screening.
Screening of the mTFP tagged *Chlamydomonas* cultures in the
fluences
microscope for conformation that the scFv (cetFv and humcetFv) is
produced. Experiment was successful due to confirmed florescence in
*Chlamydomonas* which was ruled out to be autofluorescence.

![](https://static.igem.wiki/teams/5758/notebook/figure8.webp)

**Figure 8:** Western Blot analysis of nine cytosolic cetFv
transformants of Chlamydomonas reinhardtii, UVM4 is the
negative control while PKL5 is the positive control.

![](https://static.igem.wiki/teams/5758/notebook/figure9.webp)

**Figure 9:** Western Blot analysis of nine cytosolic humcetFv
transformants of Chlamydomonas reinhardtii, UVM4 is the
negative control while PKL5 is the positive control.

![](https://static.igem.wiki/teams/5758/notebook/figure10.webp)

**Figure 10:** Fluorescence analysis of the mTFP construct. mTFP
and autofluorescence was screened independently and
subsequently overlayed. UVM4 was used as the negative control with a
cytosolic version and an version without the
scFv as the postive controle.

## Week 8

### (KW51, 16.12.24 – 22.12.24)

Christmas Break – no Lab work

## Week 9

### (KW52, 23.12.24 – 29.12.24)

Christmas Break – no lab work

## Week 10

### (KW1, 30.12.24 – 05.01.25)

Christmas Break – no lab work

## Week 11

### (KW 2, 06.01.25 – 12.01.25)

Organizational lab week after the long break, basic lab routine like
replenishing the agar- and TAP plates and cleaning the sterile hood,
preparation of new antibiotic stocks of the common used antibiotics.
Organization of the -20-degree freezer.

## Week 12

### (KW 3, 13.01.25 – 19.01.25)

Most of the Team was sick at home – no lab work

## Week 13

### (KW 4, 20.01.25 – 26.01.25)

First try of screening the secretory humcetFv and cetFv constructs. Due
to organizational diﬀiculties, we could not perform a TCA precipitation
from the media of the secretory transformants, which is routinely
utilized for secretory proteins. We opted to use another approach where
we grew *Chlamydomonas* transformants directly on a nitrocellulose
membrane in hope that the secreted scFv sticks to the membrane. We
called this approach a pseudo-Dot-Blot. This Method was quickly
abandoned due to rapid growth of mold on the agar plates (this was due
to the non-sterile nitrocellulose membrane).

![](https://static.igem.wiki/teams/5758/notebook/figure11.webp)

**Figure 11:** Pseudo-Dot-Blot method, Chlamydomonas could not
reach the nutrients on the 1% TAP Agar. The positve
controls did grew on 0.8% TAP Agar.

## Week 14

### (KW 5, 27.01.25 – 02.02.25)

Preparation for the TCA precipitation of the media from the diﬀerent
constructs with secretory signal. Inoculation of cCA-HA, ARS-HA and
GLE-HA in flasks. Preparation of the negative and positive control,
inoculation in flasks. Set cell number to 2 x 105 so that the
signal strength is comparable. Pouring SDS-Gels and preparing stock
solutions for Western Blotting.

## Week 15

### (KW 6, 03.02.25 – 09.02.25)

TCA precipitation of the media from the *Chlamydomonas* cultures
(cCA, ARS
and GLE) and freezing overnight. Loading of SDS gels with the
precipitated proteins and performing SDS-PAGE and subsequent Western
Blot. Results show that we can successfully detect the HA-Tag and
therefore the scFv from the Media, making it the first time that a scFv
of an Antibody is produced in *Chlamydomonas*. Positive control did
not
show up on test Western Blot. Inoculation of new positive control.

![](https://static.igem.wiki/teams/5758/notebook/figure12.webp)

**Figure 12:** Western Blot analysis of the TCA precipitated
proteins from the different secretory signal cultures.

## Week 16

### (KW 7, 10.02.25 – 16.02.25)

Performed TCA precipitation of two positive controls used regularly in
the department. Positive controls tested are Fast PETase and
Nanoluciferase. Performed SDS-PAGE and Western Blot of the precipitated
protein. Both positive controls did successfully show up during Western
Blot screening. Inoculation of more secretory cultures to screen for the
most transformant that produces the highest amount of scFv.

![](https://static.igem.wiki/teams/5758/notebook/figure13.webp)

**Figure 13:** Western Blot analysis of two positive controls,
Fast PETase and Nanoluciferase.

## Week 17

### (KW 8, 17.02.25 – 23.02.25)

Screening of more secretory Cultures. TCA precipitation of the Media and
SDS-PAGE with continued Western Blot. Design and ordering of new MoClo
part where we inserted a cleavage site after the scFv (of both cetFv and
humcetFv) to cleave oﬀ the SP20 and HA-Tag. Inoculation of more
*Chlamydomonas* culture.

![](https://static.igem.wiki/teams/5758/notebook/figure14.webp)

**Figure 14:** Western Blot analysis of the TCA precipitated
proteins from the different secretory signal cultures.

## Week 18

### (KW 9, 24.02.25 – 02.03.25)

Lv. 0 Moclo of the new HRV constructs. TWIST could only synthetize one
of the four designed HRV constructs. Sequencing of the Lv.0 part and
Lv.2 MoClo. Inoculation of *Chlamydomonas* for a comparison Western
Blot
of the diﬀerent secretion signals.

## Week 19

### (KW 10, 03.03.25 – 09.03.25)

TCA precipitation of the media from the diﬀerent secretory signals.
Performed SDS-PAGE and Western Blot of the precipitated proteins.
Western Blot shows that cCA secretory signal show the highest signal.
Unanimously decided that we will continue with the cCA cultures for
future experiments. Lv. 2 MoClo of the HRV did not work.

![](https://static.igem.wiki/teams/5758/notebook/figure15.webp)

**Figure 15:** comparison Western Blot analysis of the different
secretory signals, cCA produces the highest signal of
scFv.

## Week 20

### (KW 11, 10.03.25 – 16.03.25)

Inoculated new flasks for a second comparison Western Blot of the
secretory signals. Lv. 2 Moclo has continued problems as the transformed
*E. coli* did not grow on the Kanamycin LB plates. Pouring of new
LB
plates and testing out if the problem is caused by the TOP10 strain that
was used. Testing out other strains like DH5alpha. Set cell number of
the *Chlamydomonas* to 2 x 105 so that the signal
strength is
comparable.

## Week 21

### (KW 12, 17.03.25 – 23.03.25)

TCA precipitation of the media from the diﬀerent secretory signals for
the comparison Western Blot 2. Performed SDS-PAGE and Western Blot of
the precipitated proteins.

![](https://static.igem.wiki/teams/5758/notebook/figure16.webp)

**Figure 16:** second comparison Western Blot analysis of the
different secretory signals.

## Week 22

### (KW 13, 24.03.25 – 30.03.25)

Brainstormed new experiment ideas, decided on a volumetric experiment.
RPTU iGEM 2019 could show that protein accumulation stopped after
*Chlamydomonas* grew in more than 35 ml TAP media. Experiment was
set up
to 1 L TAP media. Inoculation of the cCA *Chlamydomonas* culture in
flask.

## Week 23

### (KW 14, 31.03.25 – 06.04.25)

Inoculation of *Chlamydomonas* cCA-HA-Tag cultures with a cell
density of
2 x 105 in Flasks of a volume ranging from 6 ml to 1 L.
Preparation to perform a PCR to remove the mNeonGreen from the HRV part
that we ordered from TWIST.

## Week 24

### (KW 15, 07.04.25 – 13.04.25)

Harvest of the Volumetric experiment media. Performed SDS-PAGE and
Western Blot of the precipitated proteins. We showed that our protein
accumulates at 1 L media which is a record in our department. Original
PCR failed, repeat with optimized parameters.

![](https://static.igem.wiki/teams/5758/notebook/figure17.webp)

**Figure 17:** volumetric Western Blot analysis to test the
accumulation of the scFv in higher amounts of TAP media.

## Week 25

### (KW 16, 14.04.25 – 20.04.25)

Inoculation of *Chlamydomonas* for a third comparison Western Blot
of the
secretory signals. Setting the cell density to 2 x 105.
Creation of a DMSO *E. coli* stock of all to this point created
parts,
storage in the -80-degree freezer and registration of the parts in the
oﬀicial department lists. Repeat of the PCR failed once again, second
repeat of the PCR.

## Week 26

### (KW 17, 21.04.25 – 27.04.25)

TCA precipitation of *Chlamydomonas* culture for third secretory
signal
comparison Western Blot. Performed SDS-PAGE and Western Blot of the
precipitated proteins. Third repeat of the PCR. Experiment was
successful this time. Send oﬀ PCR part for sanger sequencing.
Inoculation of *Chlamydomonas* culture for a second volumetric
accumulation experiment. Inoculation of *Chlamydomonas*
transformant with
HRV-mNG for fluorescent screening.

![](https://static.igem.wiki/teams/5758/notebook/figure18.webp)

**Figure 18:** third comparison Western Blot analysis of the
different secretory signals.

## Week 27

### (KW 18, 28.04.25 – 04.05.25)

TCA precipitation of *Chlamydomonas* culture for second volumetric
accumulation Western Blot. Performed SDS-PAGE and Western Blot of the
precipitated proteins. In addition to the TCA precipitation, we
conducted another approach where the proteins from the Media were
precipitated by 80% acetone. This approach would spare us to use the
highly dangerous TCA. PCR sanger sequencing showed a successful removal
of the mNG form the part. Lv. 2 MoClo was conducted and transformed into
*Chlamydomonas*.

![](https://static.igem.wiki/teams/5758/notebook/figure19.webp)

**Figure 19:** Western Blot analysis of the TCA precipitation
form three transformants from the cetFv and humcetFv
cultures.

![](https://static.igem.wiki/teams/5758/notebook/figure20.webp)

**Figure 20:** Western Blot analysis of the acetone precipitation
form three transformants from the cetFv and humcetFv
cultures.

## Week 28

### (KW 19, 05.11.25 – 11.05.25)

Screening of the HRV constructs for fluorescence. Assembly of a 10 L
batch bioreactor originally used for fermenting filamentous fungi.
Thoroughly cleaning of the bioreactor and autoclaving. Inoculation of
the bioreactor with *Chlamydomonas* UVM4.

![](https://static.igem.wiki/teams/5758/notebook/figure21.webp)

**Figure 21:** Fluorescence analysis of the mTFP construct. mTFP
and autofluorescence was screened independently
and subsequently overlayed. UVM4 was used as the negative
control.

![](https://static.igem.wiki/teams/5758/notebook/figure22.webp)

**Figure 22:** 10L batch bioreactor previously used to ferment
filamentous fungi.

## Week 29

### (KW 20, 12.05.25 – 18.05.25)

Planning of new experiment where we test at what timepoint we can detect
the accumulation of the protein in the media. This experiment was called
ST-test (secretion-time-test). Inoculation of *Chlamydomonas* for
ST-test.
Disassembly of the 10 L batch bioreactor, the experiment shows that
*Chlamydomonas* does grow in this system. Discontinued use of the
bioreactor due to continued contamination. Assembly of another
bioreactor called BIOSTREAM2 – flatpanel bioreactor 1.8 L. Inoculation
of cCA-humcetFv *Chlamydomonas* cultures. Inoculation of
*Chlamydomonas*
cultures for third volumetric test.

![](https://static.igem.wiki/teams/5758/notebook/figure23.webp)

**Figure 23:** assembly of the called BIOSTREAM 2 – flatpanel
bioreactor 1.8 L.

## Week 30

### (KW 21, 19.05.25 – 25.05.25)

Daily acetone protein precipitation for ST-test. Aceton precipitation of
*Chlamydomonas* culture for third volumetric accumulation Western
Blot.
Performed SDS-PAGE and Western Blot of the precipitated proteins.
Flatpanel bioreactor got contaminated, removal of contents and
autoclaving for second try. Preparations to start work in the toxicology
lab.

![](https://static.igem.wiki/teams/5758/notebook/figure24.webp)

**Figure 24:** Western Blot analysis of the second volumetric
experiment to test the accumulation of the scFv in higher
amounts of TAP media.

## Week 31

### (KW 22, 26.05.25 – 01.06.25)

Aceton precipitation of *Chlamydomonas* culture for ST-test.
Performed
SDS-PAGE and Western Blot of the precipitated proteins. Second try for
the BIOSTREAM 2 bioreactor. Starting to isolate and purify the secreted
protein with IMAC using the His-Tag transformants. Started to work in
the toxicology lab to verify the function of the humcetFv in Vivo
utilizing cell culture, learned the basics of cell culture like how to
passage cells.

![](https://static.igem.wiki/teams/5758/notebook/figure25.webp)

**Figure 25:** Western Blot analysis of the ST-test from
cCA-cetfv and cCA-humcetFv culture, showing protein
accumulation starts as early as day 2.

## Week 32

### (KW 23, 02.06.25 – 08.06.25)

BIOSTREAM 2 biorector got contaminated again, removal of contents and
autoclaving for third try. SDS-PAGE and Coomassie staining of the
isolated and purified proteins from *Chlamydomonas* culture with
His-Tag
using IMAC.

![](https://static.igem.wiki/teams/5758/notebook/figure26.webp)

**Figure 26:** Coomassie gel and corresponding Western Blot
analysis of the protein purification.

## Week 33

### (KW 24, 09.06.25 – 15.06.25)

Designed new scFv parts: Pantitumumab, Matuzumab, Trastuzumab. Ordered
the new designed parts on Twist. Continued with protein purification
using IMAC. Third BIOSTREAM 2 approach did work out, culture stayed
sterile. Taking media from the bioreactor on day 7, 8 and 9 to test via
Western Blot if protein accumulates in 1.8 L TAP media.

## Week 34

### (KW 25, 16.06.25 – 22.06.25)

SDS-PAGE and Western Blot of the Aceton precipitated media from day 7, 8
and 9 of the BIOSTREAM 2 bioreator culture. Two bioreactors were running
simultaneously; one filled with TAP media and the other with HMP Media.
Continued with protein purification using IMAC. Planning of the
resazurin reduction assay in the toxicology lab to test the scFv.

![](https://static.igem.wiki/teams/5758/notebook/figure27.webp)

**Figure 27:** BIOSTREAM 2 bioreator protein precipitation from
cCA-humcetFv culture, day 7, 8 and 9 using TAP and HMP
media.

## Week 35

### (KW 26, 23.06.25 – 29.06.25)

First attempted to test our purified scFv in vitro using cell culture.
SW48 cells should react to a treatment with the scFv whereas the
negative control HT29 should not show any reaction. First attempted of
the resazurin reduction assay did not show any eﬀects. Protein
purification is continued to treat the cells with a higher dosage.

![](https://static.igem.wiki/teams/5758/notebook/figure28.webp)

**Figure 28:** 400x zoom of the HT29 cells (left) and SW48 cells
(right)

![](https://static.igem.wiki/teams/5758/notebook/figure29.webp)

**Figure 29:** schematic overview of the treatment plan of the
HT29 and SW48 cells.

## Week 36

### (KW 27, 30.06.25 – 06.07.25)

Continued with protein purification using IMAC. Planning of an
experiment doubt pulldown-assay where we can show that the scFv can bind
the target protein by using the target protein, EGFR with an added
His-Tag which will bind during IMAC.

## Week 37

### (KW 28, 07.07.25 – 13.07.25)

Continued with protein purification using IMAC. Archived the highest
protein yield thus far with 49 µg/ml.

![](https://static.igem.wiki/teams/5758/notebook/figure30.webp)

**Figure 30:** Coomassie gel and corresponding Western Blot
analysis of the protein purification.

## Week 38

### (KW29, 14.07.25 – 20.07.25)

Lv. 0 MoClo of the new scFv Pantitumumab, Matuzumab, Trastuzumab failed.
First attempts of the pulldown assay to test the binding capacity of the
scFv (humcetFv), experiment was not successful as the negative control
showed a signal. Continued with protein purification using IMAC.

![](https://static.igem.wiki/teams/5758/notebook/figure31.webp)

**Figure 31:** Western Blot analysis of the first attempt from
the pulldown assay to show that the produced scFv can bind
its target protein (EGFR)

## Week 39

### (KW 30, 21.07.25 – 27.07.25)

Continued with protein purification using IMAC. Lv. 0 MoClo of scFv
Pantitumumab, Matuzumab, Trastuzumab failed again, after careful
inspection it was discovered that there was a mistake that happened
during the ordering of the parts. Parts were ordered again.

## Week 40

### (KW 31, 28.07.25 – 03.08.25)

Continued with protein purification using IMAC. Optimization of the
pulldown assay to show that the scFv binds the target protein. Lv. 0
MoClo of scFv Pantitumumab, Matuzumab, Trastuzumab was successful. Lv 2.
MoClo of scFv Pantitumumab, Matuzumab, Trastuzumab.

## Week 41

### (KW 32, 04.08.25 – 10.08.25)

Continued with protein purification using IMAC. Second attempt for the
pulldown assay with optimized variables. Planning of the remaining time
in the iGEM competition, it was decided that some experiments should be
redone to achieve cleaner results that we can showcase.

![](https://static.igem.wiki/teams/5758/notebook/figure32.webp)

**Figure 32:** Western Blot analysis of the second attempt from
the pulldown assay to show that the produced scFv can
bind its target protein (EGFR). The negative control Luciferase was
positive.

## Week 42

### (KW 33, 11.08.25 – 17.08.25)

Final pulldown assay with improved parameters, the negative control was
changed to fastPETase as luciferase showed aﬀinity to the nickel beads.
Results of the pulldown assay confirm a binding capacity of the scFv to
the target protein EGFR. Transformation of cCA-cetFv and cCA-humcetFv
construct into *Chlamydomonas* strain UVM11CW and ClipUVM. Reapeat
of the
TCA vs. Aceton Western Blot and comparison Blot of the secretory
signals. Transformation of Lv. 2 MoClo scFv Pantitumumab, Matuzumab,
Trastuzumab into *Chlamydomonas*.

![](https://static.igem.wiki/teams/5758/notebook/figure33.webp)

**Figure 33:** Western Blot analysis of the pulldown assay,
showing that our cetFv and humcetFv binds the EGFR target
protein, whereas the negative control PETase does not.

## Week 43

### (KW 34, 18.08.25 – 24.08.25)

Last attempt in the toxicology lab to test the in vitro capacity of the
produced and purified scFv. Resazurin reduction assay did not give any
valuable data, even when the scFv dose was increased. The in vitro
assays did not deliver negative results because the positive control
(commercial cetuximab) also did not show any eﬀects on the cells.
Pharmacological inhibitor did show slight eﬀects at the highest dosage.
The in vitro experiments are deemed improvable. SDS-PAGE and Western
Blot of TCA vs. Aceton Western Blot and comparison Blot of the secretory
signals.

![](https://static.igem.wiki/teams/5758/notebook/figure34.webp)

**Figure 34:** final comparison of the secretory signals, Western
Blot analysis for the WIKI

![](https://static.igem.wiki/teams/5758/notebook/figure35.webp)

**Figure 35:** Final Western Blot analysis of the TCA vs. acetone
precipitation for the WIKI

## Week 44

### (KW 35, 25.08.25 – 31.08.25)

Final Week in the Lab. SDS-PAGE and Western Blot of the Aceton
precipitation from the scFv Pantitumumab, Matuzumab, Trastuzumab
*Chlamydomonas* transformants. Pantitumumab and Matuzumab did not
show a
signal on the Western Blot. SDS-PAGE and Western Blot of the cCA-cetFv
and cCA-humcetFv *Chlamydomonas* strain UVM11CW and ClipUVM
transformants.

![](https://static.igem.wiki/teams/5758/notebook/figure36.webp)

**Figure 36:** Western Blot analysis of the Trastuzumab
transformants, confirming that the protein gets secreted into the
medium.

![](https://static.igem.wiki/teams/5758/notebook/figure37.webp)

**Figure 37:** Western Blot analysis of the CLiPUVM transformants
with cetFv and humcetFv

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# Protocols, media and devices we used throughout the iGEM project

## Assays

## TCA vs Acetone Precipitation

This protocol is to test whether Acetone or TCA is a more suitable
reagent for precipitating
secreted protein.

1. A preculture of one HA-protein tagged secreting Chlamydomonas
   culture, a negative
   control (e.g., UVM4) and a positive control (L5) were prepared in
   TAP-NH₄ medium.
   Cultures were incubated under continuous light (70 μmol photons
   m⁻²s⁻¹) with orbital
   shaking at 120 rpm for seven days.
2. Cell density was determined and the HA-protein tagged secreting
   Chlamydomonas strain
   was diluted into 2 Erlenmeyer flasks to a density of 2×10⁵ cells/ml.
   Two negative controls
   and one positive control were included.
3. After six days, the cultures were harvested and 5 technical
   replicates were precipitated, one
   half with [acetone](#acetone) and the other half with [TCA](#TCA).
4. 20 μl of SDS sample from the acetone-precipitated protein and ~4.1
   μl from the TCA
   precipitated protein sample were loaded onto an SDS-PAGE gel to
   ensure correct ratio of
   protein concentration.
5. [SDS-PAGE](#SDS) and [Western
   Blotting](#westernblot) were performed as described in the standard
   protocols.

![](https://static.igem.wiki/teams/5758/experiments/experiments-figure1-1.webp)

**Figure 1:** SDS-PAGE loading scheme for the TCA vs.
Acetone precipitation test. 3.5 μl PageRuler™ Plus Prestained
Protein Ladder is used as the marker. For the HA-protein tagged
secreting Chlamydomonas strain and negative
control (UVM4), 4.1 μl of TCA-precipitated sample and 20 μl of
acetone precipitated sample were loaded. PKL5 was
included as a positive control. Created with Biorender.

## Volume test

This assay is designed for the initial scale-up of protein-secreting
strains. It allows an estimation of
the culture volume that yields the highest protein amount.

1. A preculture of one HA-protein tagged secreting Chlamydomonas
   culture was prepared in
   TAP-NH₄ medium. Cultures were incubated under continuous light (70
   μmol photons
   m⁻²s⁻¹) with orbital shaking at 120 rpm for seven days.
2. Cell density was determined, and the test strain was diluted into
   1000 mL adjusting to a
   final density of 2×10⁵. The flasks from 6 mL to 400 mL were filled
   with this dilution. Dilution
   was repeated for the 1000 mL flask. Cultures were incubated again
   for six more days.
3. After six days, the supernatant was harvested, and proteins were
   [precipitated with ice-cold
   acetone](#acetone).
4. [SDS-PAGE](#SDS) and [Western
   blotting](#westernblot) were performed as described in the standard
   protocols.

![](https://static.igem.wiki/teams/5758/experiments/figure2.webp)

**Figure 2:** Volume test. **(A)** Erlenmeyer flasks
with culture volumes ranging from 6 mL to 100 mL. **(B)**
SDS-PAGE
loading scheme for the volume test. 3.5 μl PageRuler™ Plus
Prestained Protein Ladder is used as the marker. 10 μl of
acetone precipitated sample from each culture volume (6-1000 mL)
were loaded. Created with Biorender.

## ST-Test (secretion-time-Test)

This assay is designed to test the required amount of time for the
Chlamydomonas cultures to
accumulate the secreted scFv in the media. It allows optimization of
the incubation time for
subsequent experiments by determining when sufficient protein
accumulation has occurred.

1. Liquid Chlamydomonas cultures of CetFv and HumcetFv and a negative
   control (e.g. UVM4)
   were prepared. Cultures were incubated under continuous light (70
   μmol photons m⁻²s⁻¹)
   with orbital shaking at 120 rpm for several days.
2. Three 25 ml flasks were inoculated with the respective
   Chlamydomonas cultures, and the
   final cell density was adjusted to 2 × 10⁵ cells/ml.
3. Samples of the CetFv and HumcetFv cultures were harvested
   continuously for six days,
   starting at day zero after inoculation.
4. The proteins were precipitated after each harvest using the [acetone precipitation method](#acetone) and
   the acetone-precipitated proteins were frozen at –20°C.
5. The acetone precipitation protocol was continued and [SDS-PAGE](#SDS) followed by [Western
   blotting](#westernblot) was performed according to protocols.

![](https://static.igem.wiki/teams/5758/experiments/figure3-3.webp)

**Figure 3:** Schematic overview of the ST-test
workflow showing daily sampling from Chlamydomonas cultures over six
days, followed by acetone precipitation and protein analysis via
SDS-PAGE and Western blotting. Created with
Biorender.

## Pulldown-Interaction Assay

This interaction assay is designed to determine whether the
single-chain fragment can specifically
interact with its corresponding receptor epitope (e.g., EGF
receptor).

1. Liquid cultures of CetFv, HumcetFv and a negative control (e.g.,
   PETase) were prepared in
   TAP-NH₄ medium. Cultures were incubated under continuous light (70
   μmol photons
   m⁻²s⁻¹) with orbital shaking at 120 rpm for several days.
   note: test proteins must not carry a His-tag.
2. Cultures were harvested several days after stationary phase in 50
   ml Falcon tubes.
3. Cultures were centrifuged 2x at 4,000g for 4 min to harvest
   supernatant.
4. Proteins were concentrated by ammonium sulfate precipitation
   followed by dialysis in PBS
   overnight.
5. 66 μl Ni-NTA were equilibrated in PBS in a reaction tube.
6. 20 μl of the EGFR (50 ng/μl) were mixed with 300 μl of ammonium
   sulfate-concentrated
   protein and incubated for 30 min at 37°C.
7. A sample for Western blot analysis was taken, and the volume was
   adjusted to 1 ml by
   adding PBS.
8. Storage buffer was removed from Ni-NTA by plugging a white tip
   into a blue one. The
   complete sample was loaded into each Ni-NTA reaction tube and 40 μl
   of cOmplete™
   protease inhibitor were added.
9. Samples were incubated for 30 min under constant inversion, then
   placed on ice for 10 min.
10. Samples were centrifuged at 100 g for 30 sec at 4 °C.
11. A sample was collected, the supernatant was discarded, and the
    beads were washed with
    PBS and 5 mM imidazole.
12. Samples were centrifuged at 100 g for 30 sec at 4°C.
13. A sample was taken, the wash buffer was discarded, and 50 μl
    elution buffer with PBS and
    500 mM imidazole were added. Samples were incubated for 2 min.
14. Samples were centrifuged at 100 g for 30 sec at 4 °C.
15. The supernatant was collected, and all samples were [precipitated with ice-cold acetone.](#acetone)
16. [SDS-PAGE](#SDS) and [Western
    blotting](#westernblot) were performed according to standard protocols.

![](https://static.igem.wiki/teams/5758/experiments/figure4-1.webp)

**Figure 4:** Pulldown-Interaction Assay. In the first
reaction tube, EGFR and scFv interact with each other. After
incubation, the mixture is transferred into a new reaction tube with
Ni-NTA beads. The His-tagged EGFR binds to Ni-
NTA, keeping the scFv from being washed away. Created with
BioRender.

## Resazurin Assay

This assay is designed to evaluate the cytotoxic effects of various
treatments on cell viability using
a resazurin-based metabolic activity indicator.

1. 40,000 cells in triplicates per well in cell culture medium for
   the 24h incubation period and
   10,000 cells in triplicates per well in cell culture medium for the
   72h incubation period were
   plated. Cells were incubated for 24h.
2. Solutions containing the amount of drug to be tested were
   prepared. The Cetuximab, scFv,
   5-FU and saponin solutions were diluted in a volume of 700 μl for
   both plates.
3. Resazurin stock solution was prepared according to the
   manufacturer's protocol.
4. After the 24h incubation period, the medium from the wells
   containing the cells was aspirated
   and 100 μl of the respective solution were added to the wells in
   triplicates. Cells were
   incubated again, and the starting time of the treatment was noted
   down.
5. 600 μl of the prepared resazurin stock solution were mixed with
   5.4 ml of cell culture medium.
6. The medium from the cells was aspirated and 100 μl of the
   resazurin mixture were added to
   the cells. Cells were incubated for exactly 90 minutes.
7. After the incubation, cells were taken out, and the fluorescence
   intensity was measured at an
   excitation wavelength of 544 nm and an emission wavelength of 590
   nm.
8. Steps 5 – 7 were repeated for the 72h incubation plate.

![](https://static.igem.wiki/teams/5758/experiments/figure5.webp)

**Figure 5: (A)** 96-well plate after initial treatment
with the resazurin mix before the 90-minute incubation period
**(B)** 96-well plate
expectations after the 90 minutes incubation period, validating the
cytotoxic effects of the commercial cetuximab and
the scFv **(C)** results after the 90 minutes incubation period,
validating the survival of the cetuximab and scFv treated
cells.

## General Protocols

DNA Protocols

## Modular Cloning

Calculate the volume needed for a final concentration of 40 fmol for
each part. Knowing the size of the
part allows you to calculate the amount of DNA (in ng) equaling 40
fmol. (For this you can use online
tools.)
Mix the components in a small reaction tube, add the enzymes (BsaI and
T4 DNA Ligase) last. Place
tubes into a thermocycler.

| Component | Amount | Concentration | Protocol |
| --- | --- | --- | --- |
| MoClo Parts (n)  Part 1  Part 2  Part 3  ...  Destiniation vector | 1 µl | 40 fmol | 37°C 2 min  16°C 5 min  repeat 45 x  50°C 5 min  80°C 10 min  4°C ∞ |
| T4 DNA Ligase buffer | 2 µl | 1x |  |
| BsaI-HFv2® or BbsI-HF | 0.5 µl | 10 U |  |
| T4 DNA Ligase | 0.5 µl | 10 U |  |
| Milli-Q | To 20 µl | - |  |

## Transformation in *Escherichia coli*

1. Thaw One Shot® TOP10 (NEB) E. coli cells on ice.
2. Carefully pipette 10 µl of your MoClo reaction onto the cells,
   mix, and incubate for 30 min on
   ice.
   → Preheat the thermomixer to 42°C for the subsequent heat
   shock.
3. Heat shock E. coli cells for 50 s at 42°C. Recover for 5 min on
   ice.
4. Work sterile: Add 600 µl LB-medium and incubate cells for 45 min
   at 37°C in the incubator.
5. Work sterile: Spread out 300 µl onto LB-plates containing
   spectinomycin (level 0) or kanamycin (level 2).
   Let plates dry under the clean bench.
6. Incubate plates overnight at 37°C.

## Isolation of Plasmids

1. Work sterile: Prepare sterile test tubes with 5 ml LB-medium each
   containing spectinomycin (level 0)
   or kanamycin (level 2). For each transformation plate, prepare three
   tubes.
2. Take out your plates and gently pick a single white colony with a
   yellow pipette tip, which
   will then be dropped into the respective test tube. Repeat for two
   more positive colonies of
   your transformation plate.
3. Incubate test tubes overnight at 37°C.
4. Prepare Eppendorf tubes for harvesting the E. coli cultures and
   for their respective plasmids,
   which will be eluted later.
5. Harvest 2 ml by centrifugation at 4000 rpm for two minutes.
   Discard supernatant.
6. Use the plasmid prep kit provided.

* Resuspend pellet in 150 µl Buffer A1, vortex.
* Add 250 µl Buffer A2 and invert five times. Incubate for 2 min
  at RT.
* Add 350 µl Buffer A3, invert until sample turns colorless.
* Centrifuge at full speed (> 12,000 g) for 3 min. Pipette
  supernatant (750 µl) onto the column
  provided with the kit.
* Centrifuge columns at 2000 g for 30 s. Discard flow
  through.
* Add 450 µl Buffer AQ, centrifuge at full speed (> 12,000 g) for
  1 min.
* Transfer column to the prepared Eppendorf tube. Add 50 µl Buffer
  AE onto the column
  membrane, incubate at least 1 min at RT.
* Centrifuge 1 min at full speed to elute plasmids from the
  column.

7. Measure DNA concentration at the Nanodrop. Pipette 1 µl of Buffer
   AE onto the sample spot
   for blank measurement. Then pipette 1 µl of your plasmid and measure
   the DNA concentration.

## Test Digest

1. Mix components in Eppendorf tube and incubate at 37°C for 45
   min.
2. Prepare 1% agarose gels with 1x TAE buffer. Dissolve 1 g agarose
   in 100 ml 1x
   TAE. Heat solution in microwave and let cool down to 60°C. Then add
   5 µl HD Green. Pour agarose gels.
3. Add 4 µl of 6x loading dye to your test digest samples and load 10
   µl onto the agarose gel.
4. Separate DNA fragments by gel electrophoresis (110 V, 45 – 55
   min).
5. Analyze your gels using the Intas device. Compare your observed
   fragments with the
   theoretical in silico test digest.

## Nuclear Transformation via Glass Beads

Work sterile! Chlamy clean bench!

1. For each transformation process:
   Linearize 1 µg DNA in a final volume of 10 µl (1 µg DNA, 1 µl
   CutSmart buffer, 0.5 µl enzyme,
   to 10 µl with MQ)
   → Use enzyme, which only cuts the backbone of your construct (Check
   in silico)!
2. Digest/linearize your construct for 45 min at 37°C.
3. Measure cell density with the CoulterCounter
4. For each transformation 5\*107 cells are needed, calculate the
   volume according to the cell
   density of your culture. Always include a negative control without
   DNA!
5. Centrifuge cultures at 4000 rpm for 2 minutes (use sterile 50 ml
   Falcon tubes).
6. Discard supernatant and resuspend cells in 330 µl fresh TAP
   medium.
7. Add 330 µl of your cell suspension to the sterile reaction tubes
   filled with glass beads, pipette
   10 µl of the linearized DNA to the side of the tube → for the
   negative control, do not add any DNA.
8. Take two tubes and vortex for 15 s
9. Plate out 330 µl onto your plates containing the appropriate
   antibiotic (avoid glass beads on your plates!)
   → This works best if you pipette at the bottom of the tube and
   remove the remaining beads on the lid.
10. Let plates dry under the hood, seal with Parafilm and place into
    the dark overnight.
11. Place plates into the light in the next morning.
12. Wait several days until single colonies appear. Transfer single
    colonies to fresh agar plates.

## Site-Directed Mutagenisis

1. Pipette the following reagents into PCR tubes:

| Reagent | Volume |
| --- | --- |
| Q5 Buffer | 10 µl |
| dNTPs | 1 µl |
| Primer forward | 2.5 µl |
| Primer reversed | 2.5 µl |
| Template DNA | 10 µl |
| Q5 Polymerase | 0.5 µl |
| GC Enhancer | 10 µl |
| MQ | 13.5 µl |

2. Start the PCR with the following cycles

| STEP | TEMP | TIME |
| --- | --- | --- |
| Initial Denaturation | 98 °C | 30 sec |
| 10 Cycles | 98 °C  63°-72° gradient  72 °C | 07 sec  25 sec  2 min |
| 25 Cycles | 98 °C  72 °C  72 °C | 07 sec  25 sec  2 min |
| Final Extension | 72 °C | 2 min |
| Hold | 4 °C | ∞ |

## PCR Clean-Up

[NucleoSpin®
Gel and PCR Clean-up Kit and protocol by MACHEREY-NAGEL were used](https://www.mn-net.com/media/pdf/02/1a/74/Instruction-NucleoSpin-Gel-and-PCR-Clean-up.pdf)

1. Adjust DNA binding conditions

* For very small sample volumes < 30 μL adjust the volume of the
  reaction mixture to 50–100 μL with water.
* Mix 1 volume of sample with 2 volumes of Buffer NTI (e.g., mix
  100 μL PCR reaction and 200 μL Buffer NTI).

2. Bind DNA

* Place a NucleoSpin® Gel and PCR Clean-up Column into a
  Collection Tube (2 mL) and load up to 700 μL sample.
* Centrifuge for 30 s at 11,000 x g. Discard flow-through and
  place the column back into the collection tube.
* Load remaining sample if necessary and repeat the centrifugation
  step.

3. Wash silica membrane

* Add 700 μL Buffer NT3 to the NucleoSpin® Gel and PCR Clean-up
  Column. Centrifuge for 30 s at 11,000 x g. Discard flow-through
  and place the column back into the collection tube.
* Recommended: Repeat previous washing step to minimize chaotropic
  salt carry-over and improve A260/A230 values (see section 2.7 for
  detailed information).

4. Dry silica membrane

* Centrifuge for 1 min at 11,000 x g to remove Buffer NT3
  completely. Make sure the spin column does not come in contact
  with the flow-through while removing it from the centrifuge and
  the collection tube.

5. Elute DNA

* Place the NucleoSpin® Gel and PCR Clean-up Column into a new 1.5
  mL microcentrifuge tube (not provided). Add 15–30 μL of hot Buffer NE
  and incubate at 70 °C for 5 min. Centrifuge for 1 min at 11,000 x
  g

Protein Analysis & Quantification

## TCA Precipitation of Chlamydomonas Supernatant

1. Harvest 1.9 ml liquid culture of the transformants of interest in
   a reaction vessel. Culture growth time should be at least 3 days (6
   days would be optimal)
2. Centrifuge at 1,700 g for 4 min, transfer 1.8 ml supernatant into
   a new reaction vessel
3. Centrifuge again at 4,000 g for 4 min, transfer 1.7 ml supernatant
   into a new 2 ml reaction tube
4. Add 230 µl ice-cold TCA (be careful TCA is caustic!), mix your
   samples thoroughly and them on ice for 30 minutes.
5. Cetrifuge all samples at 15,000 g for 15 min at 4 °C
6. Precipitated protein now remains in the sediment. Carefully remove
   the supernatant using a white tip plugged into a blue one
7. Add 100 µl PBS onto the sediment and vortex the sample
   thoroughly
8. Add 600 µl ice-cold acetone and vortex samples thoroughly again.
   Incubate samples overnight at
   – 20 °C. (Make sure freezer is suitable for storing volatile
   substances (explosion-proof freezer)!)
9. Centrifuge samples for 5 min at 15,000 g and 4 °C.
10. Remove supernatant using a white tip plugged into a blue one.
11. Dry sediment by incubating the samples with an open lid at 37 °C
    for about 3 min. Make sure that all acetone is evaporated. Do not
    overdry the sediment!
12. Add 20 µl 1x SDS loading buffer (make sure that DTT is added to
    your SDS loading buffer)
13. Repeat the following steps multiple times:

* Vortex samples thoroughly
* Incubate samples at 95 °C for 5 min
* Vortex samples thoroughly
* Sonicate samples in water bath for 5 min
* Inspect if pellet is fully resuspended

14. Carefully load 10-12 µl onto an SDS gel (high viscosity!) and
    perform SDS-PAGE usual

## Acetone Precipitation of Chlamydomonas Supernatant

1. Harvest 800 µl liquid culture of the transformants of interest in
   a reaction vessel. Culture growth time should be at least 3 days (6
   days would be optimal).
2. Centrifuge at 1,700 g for 4 min, transfer 700 µl supernatant into
   a new reaction vessel.
3. Centrifuge again at 4.000 g for 4 min, transfer 350 µl supernatant
   into a new 2 ml reaction tube.
4. Add 1575 µl ice cold acetone, mix your samples thoroughly and
   incubate samples overnight at – 20 °C. (Make sure freezer is
   suitable for storing volatile substances (explosion-proof
   freezer)!)
5. Cetrifuge samples for 5 min at 15,000 g and 4 °C.
6. Remove supernatant using a white tip plugged into a blue one.
7. Dry sediment by incubating the samples with an open lid at 37 °C
   for about 3 min. Make sure that all acetone is evaporated. Do not
   over dry the sediment!
8. Add 20 µl 1x SDS loading buffer (make sure that DTT is added to
   your SDS loading buffer)
9. Repeat the following steps multiple times:

* Vortex samples thoroughly
* Incubate samples at 95 °C for 5 min
* Vortex samples thoroughly
* Sonicate samples in water bath for 5 min
* Inspect if pellet is fully resuspended

10. Carefully load 10-12 µl onto a SDS gel (high viscosity!) and
    perform SDS-PAGE usual

## SDS-Gel Electrophoresis

Work with gloves (unpolymerized acrylamide is toxic and keratin gives
contaminating bands)!

1. Wash glass plates thoroughly with sponge and soap, rinse well with
   VE water and wipe with
   70% ethanol. Assemble glass plates into gel pouring device.
2. Prepare solutions for 4x separation and 4x stacking gels in 50-ml
   Falcons (don’t add APS and TEMED).
3. Add APS and TEMED to separating gel solution and mix
   well.
4. Pour gel solution between the glass plates in the pouring device
   and overlay with isopropanol
   using a Pasteur pipette. Let the gel polymerize for 45 - 60
   min.
5. Decant isopropanol and rinse gels thoroughly with distilled water
   and remove remaining water
   on top of the gel with Whatman paper (avoid touching the gel while
   doing this!).
6. Add APS and TEMED to the stacking gel solution and mix well.
7. Pour solution onto the separating gel and slide-in combs;
   avoid bubbles!
8. Let the gel polymerize for 30 - 45 min. (Optionally seal the gels
   in a plastic bag/wrap and store
   at 4° until use (possible for several days).)
9. Carefully remove combs and mark the pockets on the outer glass
   plate.
10. Assemble glass plates with gels into the running device and place
    it into the running chamber.
11. Fill-in electrode buffer into the interior chamber until the
    buffer level is between outer and inner glass plates; pour remaining
    buffer into the outer chamber.
12. Load 10-12 µl of the samples using a 20-µl pipette and load 3 µl
    of protein marker (M).
13. Conduct electrophoresis at 150 V for ~60 min;
    in the meanwhile, prepare Whatman paper and nitrocellulose membranes
    for blotting.

## Western Blotting

**Work with gloves!**

1. Cut 10 Whatman papers and one Nitrocellulose membrane to 9 x 6 cm
   (this stuff is expensive,
   avoid wasting; never touch the membrane without gloves!); label
   membrane with a pencil at
   the upper right corner, this will become the side to which proteins
   are blotted.
2. In one corner of a large glass plate, soak 3 Whatman papers with
   buffer T3 and squeeze out
   bubbles by rolling over the stack with a test tube; add a little
   more buffer 3 and place the three
   filter papers onto the anode of a semi-dry blotting device.
3. In another corner of the glass plate, soak 2 Whatman papers with
   buffer T1 and squeeze out
   bubbles with a fresh test tube; add a little more buffer T1.
4. Place gel left side right and without bubbles onto the two Whatman
   papers.
5. Soak Nitrocellulose membrane in buffer T2 and place it onto the
   gel such that the pencil mark
   faces the gel.
6. Soak 2 more Whatman papers in buffer T2 and place them onto the
   membrane; squeeze out
   bubbles with a test tube starting from the middle into all
   directions (don’t apply too much
   pressure!).
7. Take the entire stack, turn it upside-down and place it onto the
   three Whatman papers soaked
   with buffer T3 that are already on the anode.
8. In another corner of the glass plate, soak 3 Whatman papers with
   buffer T1 and squeeze out
   bubbles with a fresh test tube; then place the three papers onto the
   sandwich on the anode.
9. Close blotting device by placing the cathode on top and conduct
   transfer for 60 min by applying
   a current of 0.8 mA/cm2 per gel surface.

## Ponceau-Staining

**Work with gloves!**

1. Disassemble the sandwich after the transfer and incubate membrane
   for 1 min in Ponceau–S
   solution (touch membrane only with forceps! Be careful, it is very
   fragile!)
2. Pour the Ponceau solution back into its container (it is re-used
   several times) and wash
   membrane with distilled water until bands appear brightly without
   background.
3. Take an image of the membrane using the FUSION device.
4. Allow the membrane to dry on a Whatman paper.

## ECL Detection

1. Blocking: Incubate the membrane in a plastic container with 15 ml
   PBS-TM for 30 min; shake
   gently and ensure that the membrane is always covered with
   solution.
2. Remove blocking solution.
3. Primary antibody: Depending on the antibody titer and the
   abundance of the target protein,
   dilute primary antibody by 1:1000 to 1:5000 in 15 ml PBS-TM and
   incubate the membrane with
   the solution for 1 h with gentle shaking.
4. Collect the primary antibody solution in a Falcon tube.
5. Rinse membrane twice with a little PBS-T and wash membrane 3x 5
   min with 15 ml PBS-T.
6. Secondary antibody: Dilute secondary antibody (usually goat
   anti-rabbit coupled to horseradish
   peroxidase) at a 1:10000 dilution in 15 ml PBS-TM and incubate the
   membrane with this solution
   for 1 h with gentle shaking.
7. Discard the secondary antibody solution.
8. Rinse membrane twice with a little PBS-T and wash membrane 3x 5
   min with 15 ml PBS-T.
9. Cut Whatman paper to a size a little larger than the membrane and
   place it onto the FUSION
   detection tray.
10. For each mini-gel blot mix 2 ml of ECL solution I and 2 ml of ECL
    solution II in a test tube and
    vortex well (careful: do not cross-contaminate ECL solutions, this
    will destroy them). Work with
    gloves.
11. Distribute the mixed ECL solutions evenly across the Whatman
    filter paper; squeeze out bubbles
    with a test tube.
12. Place membranes with the protein side up onto the Whatman filter
    paper.
13. Detect the chemiluminescence signal with the FUSION device.

## Coomassie Staining

1. Take the gel out of the glass plates and place it for 2 min in a
   clean tray in destaining solution on
   a shaker.
2. Remove the destainer, then add the staining solution covering the
   gel and place it on a shaker
   for ca. 1 h.
3. Remove the staining solution and add destainer, shake for 5 min
   and replace the destainer (you
   can put some sheets of paper tissue next to the gel into the
   destainer; this will absorb the
   Coomassie faster).
4. Stop when the bands are clearly visible, and the background color
   is faint (takes 1-4 h).

Protein Precipitation & Purification

## Ammonium Sulfate Precipitation for Protein Sample Concentration

*Avanti centrifuge*

1. Transfer the supernatant into centrifuge bottles. Use the scale to
   balance them precisely!
2. Centrifuge using the Avanti centrifuge at 4,000 × g for 5 minutes
   (Set the centrifuge to “Yes” and ID: 14. Choose the correct rotor.
   If necessary, remove any liquid from the rotor.)
3. Open the centrifuge using the foot pedal. Transfer the supernatant
   to a new centrifuge bottle and repeat step 2.
4. 4. Transfer the supernatant into a large measuring cylinder.
   Add 53.3 g of ammonium sulfate per 100 ml of solution and stir for
   approx. 30 minutes.
5. Fill the liquid into centrifuge bottles (balance carefully!) and
   centrifuge at 12,000 × g for 15 minutes (Grease the rim of the
   centrifuge lid to ensure a vacuum seal.)
6. 6. After centrifugation, discard the supernatant.
   Resuspend the pellet in approximately 1% of the original volume
   using Equilibrator buffer.

## Dialysis

1. Prepare 5 liters of Equilibrator (or PBS) buffer in a large
   measuring cylinder:

* 12.11 g Tris (0.02 M)
* 87.66 g NaCl (0.3 M)
* Add 4.5 liters of deionized water, adjust pH to 8 with HCl
  (32%), then top up to 5 liters
  **(It is recommended to dilute HCl before use (e.g., 1:2).
  Caution: corrosive fumes –
  work under a fume hood! Wear lab coat, gloves, and safety
  goggles!)**

2. Fill the resuspended protein pellet into a dialysis tubing (don’t
   forget to clamp it!).
3. Place 800 ml of Equilibrator buffer into a new beaker.
   Immerse the sealed dialysis tubing under gentle stirring and
   incubate for a few hours at 4°C.
4. Afterwards, transfer the dialysis tubing into the remaining ~4.2
   liters of buffer and incubate overnight at **4°C** (*Very
   gentle* stirring can help.)
5. The next day, open the dialysis tubing, transfer the contents into
   Falcon tubes, and begin purification if necessary.

## Protein Purification

1. Add ~1.5 ml - 2 ml of Nickel-NTA beads to the column (ensure the
   resin is well resuspended before loading)
2. Prepare the protein sample: either by ammonium sulfate
   precipitation followed by dialysis, or by harvesting the liquid
   culture of interest and centrifuging at 4.000 g for 5 min. Transfer
   the supernatant to a new vessel and repeat this step. Make sure to
   add NaCl to a final concentration of 300 mM.
3. Remove most of the storage buffer from the Ni-NTA. Add 10 bed
   volumes (bv) equilibration buffer and allow the majority of the
   fluid to drip out of the column.
4. Add dialyzed or centrifuged supernatant to the column and allow it
   to flow through by gravity.
5. Add ~10 bv Wash buffer and allow it to drain by gravity.
6. Elute the bound protein by adding 2 ml Elution buffer. Incubate
   the resin with the elution buffer for 1-2 min. collect the complete
   eluate in two 1.5 ml reaction vessels. Repeat the elution step a
   second time.

## Wash and Regeneration of Nickel-NTA

[protocol
from cube-biotech](https://cube-biotech.com/media/09/7f/2d/1664969698/His-Washing%20Regeneration%20NTA%20IDA.pdf)

1. Remove the majority of the fluid in the column containing the
   Ni-NTA or Ni-IDA matrix. Add 10 bv dd water and allow the majority
   of the volume to drip out of the column.
2. Add 10 bv 100 mM EDTA to the column and allow the entire volume to
   flow through the matrix.
   Rinse the column again with 10 bv dd water.
3. Add 10 bv NaOH Wash Buffer to the column and allow the entire
   volume to flow through the matrix.
4. Rinse the column with 10 bv dd water.
5. Add 10 bv 10m M NiSO4 to recharge the matrix. Allow the volume to
   drip through the column by gravity.
6. Rinse the column with 5 bv dd water
7. Add 10 bv of 20% (v/v) ethanol and allow the majority of the
   volume to drip out of the column. The matrix is now ready to be
   re-used.

## Media

*E. coli* Media

## LB Medium

| Ingredient | For 1 Liter |
| --- | --- |
| NaCl | 10 g |
| Bacto yeast extract | 5 g |
| Bacto trypton | 10 g |
| ddH2O | Fill up to 1 L |
| For Agar plates: add 13 g Agar-Agar (1.3%) | |

## Ingredients of LB for Agar Plates for MoClo Transformation

| Level | Ingredient | Stock concentration | Solvent | Concentration in medium |
| --- | --- | --- | --- | --- |
| L0 | Spectinomycin IPTG X-Gal | 100 mg/ml 0.5 M 40 mg/ml | ddH2O ddH2O Dimethylformamide | 100 µg/ml 0.5 mM 40 µg/ml |
| L2 | Kanamycin | 50 mg/ml | ddH2O | 50 µg/ml |

## Antibiotics for *E. coli* Cuture Medium

| Level | Antibiotic | Solvent | Stock concentration | Concentration in medium |
| --- | --- | --- | --- | --- |
| 0 | Spectinomycin | ddH2O | 100 mg/ml | 100 µg/ml |
| 2 | Kanamycin | ddH2O | 50 mg/ml | 50 µg/ml |

*Chlamydomonas* Medium

## TAP-NH4 Medium

| Ingredient | For 1 l | For 5 l |
| --- | --- | --- |
| Tris | 2.42 g | 12.1 g |
| 4xBeijerinck-NH4 | 25 ml | 125 ml |
| 1M posphate-buffer ph7 | 1 ml | 5 ml |
| „Special K“ Trace elements: | | || EDTA-Na2 | 1 ml | 5 ml |
| (NH4)6Mo7O24 | 1 ml | 5 ml |
| Na2SeO3 | 1 ml | 5 ml |
| Zn EDTA | 1 ml | 5 ml |
| Mn EDTA | 1 ml | 5 ml |
| Fe EDTA | 1ml | 5 ml |
| Cu EDTA | 1 ml | 5 ml |
| Acetic acid | 1 ml | 5 ml |
| ddH2O | Fill up to 1 l | Fill up to 5 l |
| Adjust with acetic acid to pH 7 | | |
| For Agar plates: add 13 g Agar-Agar per liter (1,3 %) | | |

## HMP-Medium

| Ingredient | For 1 l | For 5 l |
| --- | --- | --- |
| HEPES | 4,77 g | 23.38 g |
| 4xBeijerinck-NH4 | 25 ml | 125 ml |
| 1M posphate-buffer ph7 | 1 ml | 5 ml |
| „Special K“ Trace elements: | | || EDTA-Na2 | 1 ml | 5 ml |
| (NH4)6Mo7O24 | 1 ml | 5 ml |
| Na2SeO3 | 1 ml | 5 ml |
| Zn EDTA | 1 ml | 5 ml |
| Mn EDTA | 1 ml | 5 ml |
| Fe EDTA | 1ml | 5 ml |
| Cu EDTA | 1 ml | 5 ml |
| Acetic acid | 1 ml | 5 ml |
| ddH2O | Fill up to 1 l | Fill up to 5 l |
| Adjust with KOH to pH 7 | | |
| For Agar plates: add 13 g Agar-Agar per liter (1,3 %) | | |

## Antibiotics for Chlamydomonas Agar Plates

|  | Antibiotic | Solvent | Stock concentration | Concentration in medium |
| --- | --- | --- | --- | --- |
| Spec plates | Spectinomycin | ddH2O | 100 mg/ml | 100 µg/ml |
| Amp plates | Ampicillin | ddH2O | 100 mg/ml | 100 µg/ml |
| Decontamination plates | Ampicillin | ddH2O | 500 mg/ml | 500 µg/ml |
| Cetotaxime | ddH2O | 100 mg/ml | 100 µg/ml |
| Carbendazime | 50% Ethanol | 40 mg/ml | 40 µg/ml |

General Buffers

## Equilibrator

| Reagent | Final Concentration | For 500ml |
| 3 M NaCl | 0,3 M | 50 ml |
| 0,1 M Tris pH 8 | 0,02 M | 100 ml |
| ddH2O | / | 350 ml |

## Wash Buffer

| Reagent | Final Concentration | For 500 ml |
| --- | --- | --- |
| 3M NaCl | 0.3 M | 50 ml |
| 0.1 M Tris pH 8 | 0.02 M | 100 ml |
| 2M Imidazole | 0.02 M | 5 ml |
| ddH2O | / | 345 ml |

## Elution Buffer

| Reagent | Final Concentration | For 100 ml |
| --- | --- | --- |
| 3 M NaCl | 300 mM | 50 ml |
| 0,1 M Tris pH 8 | 20 mM | 100 ml |
| 2 M Imidazol | 500 mM | 5 ml |
| ddH2O | / | 345 ml |

## EDTA Buffer

| Reagent | Final Concentration | For 250 ml |
| --- | --- | --- |
| Disodium EDTA dihydrate | 100 mM | 9.3 g |
| ddH2O | / | Fill up to 250 ml |

## NaOH Wash Buffer

| Reagent | Final Concentration | For 250 ml |
| --- | --- | --- |
| 5 M NaCl | 2 M | 100 ml |
| 1 M NaOH | 500 mM | 125 ml |
| ddH2O | / | 25 ml |

## NiSO4-Buffer

| Reagent | Final Concentration | For 250 ml |
| --- | --- | --- |
| NiSO4 \* 6xddH2O | 10 mM | 0.657 g |
| ddH2O | / | Fill up to 250 ml |

## TAE Buffer

| 50x TAE buffer | 1x TAE buffer |
| --- | --- |
| 242 g Tris  57.1 ml acetic acid  100 ml 0.5 M EDTA pH 8 | **20 ml 50 x TAE** |
| Fill up to 1 l with ddH2O | |

## Stacking & Separation Gels

| Reagent | Stacking gel | Separation gel |
| --- | --- | --- |
| 1.5 M Tris-HCL pH 8.8 | - | 5 ml |
| 0.5 M Tris-HCL pH 6.8 | 2.5 ml | - |
| 30% acrylamide | 1.1 ml | 6.8 ml |
| ddH2O | 6.1 ml | 8 ml |
| 20% SDS | 50 µl | 100 µl |
| 0.5 M EDTA pH 8 | 50 µl | 100 µl |
| APS (100 mg/ml) | 100 µl | 200 µl |
| THEMED | 20 µl | 12 µl |
| **final** | **10 ml** | **20 ml** |

## 1x Laemmli Running Buffer

| Reagent | Final Concentration | For 2 l |
| --- | --- | --- |
| 5x SDS-Page Stock | 24.8 mM Tris  134.2 mM Glycin | 400 ml |
| 20% SDS | 0.1 % | 10 ml |
| 500 mM EDTA | 1 mM | 4 ml |
| ddH2O | / | Fill up to 2 L |

## 4x Loading buffer

|  |  |
| --- | --- |
| Weigh in: | **0.6 g SDS pellets  4 g sucrose** |
| Add: 0.5 M Tris-HCl pH 6.8 | 5 ml |
| Fill up to 8 ml with | ddH2O |
| Bevor use: add 20% 1M DTT | |

## Destaining Solutions

| Reagent | For 1 l |
| --- | --- |
| 40 % Ethanol | 400 ml |
| 7 % Ac-COOH | 70 ml |
| Fill up to 1 l | |

## Staining Solution

Destaining solution + 0.125 % Coomassie brilliant blue
R-250

## Blotting Solutions

| Reagent | Buffer T3 | Buffer T2 |
| --- | --- | --- |
| ddH2O | 500 ml | 775 ml |
| 1 M Tris-HCl pH 10.4 | 300 ml | 25 ml |
| Isopropanol | 200 ml | 200 ml |

| Buffer T1 | |
| --- | --- |
| Buffer T2 | 100 ml |
| ε-aminocaproic acid | 525 mg |

## Blocking & Washing Buffer

| Reagent | 10x PBS | PBS-T | PBS-TM |
| --- | --- | --- | --- |
| NaCl  KCl  Na2HPO4  KH2PO4 | 80 g  2 g   14.6 g  2 g | 100 ml 10x PBS | 100 ml PBS-T |
| 20% Tween-20 | - | 5 ml | - |
| Milk powder | - | - | 3 g |
| ddH2O | Fill up to 1 l | 895 ml | - |

## ECL Detection Buffer

| Reagent | ECL solution I | ECL solution II |
| --- | --- | --- |
| 1 M Tris-HCl pH 8.5 | 5 ml | 5 ml |
| 250 mM luminol in DMSO | 500 µl | / |
| 90 mM p-coumaric acid in DMSO | 220 µl | / |
| 30 % H2O | / | 30.5 µl |
| Final | 50 ml | 50 ml |

---

## Premixed Buffers, Enzymes, etc.

|  |  |
| --- | --- |
| HDGreen Plus DNA Stain | INTAS science imaging |
| rCutSmartTM Buffer | New England Biolabs inc. |
| Gel Loading Dye Purple (6x) | New England Biolabs inc. |
| Quick-Load® Purple 1kb Plus Ladder | New England Biolabs inc. |
| EcoRV-HF® | New England Biolabs inc. |
| BsaI-HFv2® | New England Biolabs inc. |
| Hi-T4TM DNA Ligase | New England Biolabs inc. |
| 10x Buffer for T4 DNA Ligase with 10 mM ATP | New England Biolabs inc. |
| BamHI-HF ® | New England Biolabs inc. |
| BbsI-HF | New England Biolabs inc. |
| SapI | New England Biolabs inc. |
| Q5 High GC Enhancer | New England Biolabs inc. |
| Q5 DNA Polymerase | New England Biolabs inc. |
| Q5 Reaction Buffer | New England Biolabs inc. |
| PageRuler™ Plus Prestained Protein Ladder | Thermo Scientific |
| 10 mM dNTP Mix | Thermo Scientific |
| KpnI | Fermentas |
| His Epitope Tag Mouse mAB igG1 | Dianova |

## Devices

|  |  |
| --- | --- |
| Primus 96 Plus 96-Well PCR Thermocycler | MWG Biotech AG |
| FastGene Ultra Cycler Gradient (96-well) | NIPPON Genetics |
| BX53 microscope | Olympus |
| U-HSCBM | Olympus |
| X-Cite 120PC Q | Lumen Dynamics |
| Ultrospec 2100 pro | Amersham Biosciences |
| PJ3000 scale | Mettler |
| Toledo FE20 pH meter | Mettler |
| AE 160 precision scale | Mettler |
| NanoDrop 2000 Spectrophotometer | Thermo scientific |
| Gelimager Chemostar ECL | Intas |
| Pioneer PX323/E precision scale | Ohaus |
| Transferpette S | Brand |
| Unimax 1010 shaker | heidolph |
| Vortexer VX100 | Labnet |
| Small shaker VM3 | CAT |
| Centrifuge 5810 R | Eppendorf |
| Centrifuge 5910 R | Eppendorf |
| Dri-Block® FDB03DD | Techne |
| Fusion-FX7 Imaging system | Vilber |
| Electrophoresis Power Supply – EPS 301 | GE Healthcare |
| RCT magnetic stirrer | IKA |
| HERAsafe® KSP18 | Kendro |
| Sorvall Lynx 6000 Centrifuge | Thermo scientific |
| Avanti J-26S XP Centrifuge | Beckman Coulter |
| Solaris Shaker | Thermo scientific |
| SONOREX Super RK 514 BH | Bandelin |
| CERTOMAT® RM | Sartorius stedim biotech |
| SCANLAF MARS | LaboGene |
| Celldrop FL Fluorescence Cell Counter | DeNovix® |
| Micro centrifuge | Carl Roth |
| Drying oven | memmert |

{% endblock %}

{% extends "layout.html" %}
{% block extra\_css %}
{% endblock %}
{% block title %}Results{% endblock %}
{% block page\_content %}

* ## [Getting Started](#GettingS)
* ## [Optimization of Methods](#OptiMeth)
* ## [Proof of concept](#proof)
* ## [Outlook](#outlook)
* ## [Conclusions](#conclusion)

# Results

Our goal was to set a new milestone in cancer treatment, by demonstrating
that the unicellular
microalga *Chlamydomonas reinhardtii* (*Chlamydomonas*) is capable
of producing
functional
therapeutic antibodies. As a green alga, *Chlamydomonas* offers unique
sustainability advantages
for biopharmaceutical protein production: it harnesses light energy through
photosynthesis,
eliminating the need for expensive organic carbon sources while actively
reducing CO₂ emissions
during cultivation. Beyond its ecological benefits, cultivation is
straightforward, cost-effective, and
potentially scalable when grown in inexpensive media. However, since the use
of *Chlamydomonas*
for large-scale protein production is still under active research, we
focused our efforts on proving
the production and functionality of therapeutic single-chain antibodies in
*Chlamydomonas*.

---

## Getting started

### Design & Modular Cloning

In order to design our first constructs, we first had to select the coding
sequences. We decided to
use the sequence of the single-chain variable fragment (scFv) of the
Cetuximab monoclonal
antibody provided by the iGEM 2015 team Apollo [[1]](https://parts.igem.org/Part%3ABBa_K1694004) (CetFv), as well
as a humanized sequence of
the Cetuximab scFv described by Banisadr et al. in 2018 [[2]](https://doi.org/10.1080/21645515.2017.1407482) (HumcetFv).
We reverse-translated
these sequences with optimal *Chlamydomonas* codon usage and inserted
native
introns from
RBCS2 approximately every 150-200 nucleotides to ensure efficient transgene
expression[[3]](https://doi.org/10.1093/nar/gky532).
Finally, we added BbsI restriction sites to enable Modular Cloning.

After obtaining the two sequences from TWIST, we designed our first Level 2
constructs to answer
the following questions:

1. Can we produce and secrete scFvs in *Chlamydomonas*?

2. Can scFv production and maturation be tracked via fluorescence in
*Chlamydomonas*?

We also designed Level 2 constructs containing an 8xHis tag for
purification.

![](https://static.igem.wiki/teams/5758/results/figure1-7.avif)

**Figure 1:** Level 2 MoClo constructs for expression in
*Chlamydomonas*, labeled from 1 to 20. All constructs contain a
spectinomycin resistance cassette as well as the strong HSP70A-RBCS2 (AR)
fusion promoter. Depending on the
design, different secretion signals were included (like cCA, ARS2, GLE or
none), followed by either CetFv, HumcetFv or
a fluorescent protein (mTFP, mVenus). C-terminal tags were incorporated
for specific purposes such as Western blot
detection (HA) or fluorescence monitoring, glycosylation (SP20) or protein
purification (His tag). All constructs were
terminated by the RPL23 terminator.

The constructs shown in **Fig. 1** set the groundwork for our research.
To
address our first question
of whether we can produce and secrete scFvs in *Chlamydomonas*, we
transformed
Level 2 constructs 9-cCA-CetFv-SP20, 3xHA (SH) and 10-cCA-HumCetFv-SH (**see Fig.
1**)
into
*Chlamydomonas*, grew individual transformants in liquid culture,
precipitated
proteins in the culture
medium with acetone and analyzed them by SDS-PAGE and Western blotting using
an anti-HA
antibody. This demonstrated for the first time the production of scFv in
*Chlamydomonas*, establishing
the basis for further experiments.

![](https://static.igem.wiki/teams/5758/results/figure2.webp)

**Figure 2:** Western Blot screening of *Chlamydomonas*
transformants
probed with anti-HA primary antibody.
Transformants containing construct 9-cCA-CetFv-SH **(A)** and
10-cCA-HumcetFv-SH **(B)** analyzed for the presence
of secreted recombinant proteins in the culture medium. Lane numbers
correspond to the individual transformants
tested.

As shown in **Fig. 2**, a clear HA-signal at approximately 55 kDa was
detected,
which corresponds
to a larger than predicted molecular mass of 23 kDa. This shift can be
explained by glycosylation
of the SP20 module during secretion, which increases the apparent molecular
mass of the protein
and supports efficient secretion. So, we are confident that the detected
proteins in **Fig. 2** indeed
are our scFvs, confirming successful scFv production in
*Chlamydomonas*. The
bands indicated
below could be due to partially glycosylated or degraded scFv.

### Screening via Fluorescence

While screening for transformants producing our scFvs by Western blotting,
we also aimed to detect
the humanized Cetuximab scFv fused with mTFP via fluorescence microscopy to
address our
second question. Successful detection would provide the first visualization
of a scFv produced in
*Chlamydomonas*.

For this experiment, transformants containing constructs 6-8 (**see Fig.
1**)
were grown in a 24-well
plate alongside a negative control without expression of any fluorescent
protein and positive
controls accumulating mTFP in the cytosol or chloroplast.

![](https://static.igem.wiki/teams/5758/results/figure3.webp)

**Figure 3:** Brightfield, mTFP and chlorophyll
autofluorescence
imaging of *Chlamydomonas* transformants using an
Olympus BX53 epifluorescence microscope at 1000x magnification. Samples
include a negative control (recipient
strain), two positive controls (either accumulating mTFP in the
chloroplast or the cytosol, construct 6), a mTFP secreting
transformant (construct 7) and a transformant secreting HumcetFv fused
C-terminally with mTFP (construct 8).
Chlorophyll autofluorescence was detected at 440 nm, while mTFP
fluorescence was recorded at 270 nm. Both mTFP
and chlorophyll autofluorescence channels were merged for colocalization
analysis.

As shown in **Fig. 3**, only very little fluorescence leaks into the
mTFP
channel in the negative control.
However, chlorophyll autofluorescence was clearly visible at 440 nm. The
fluorescence from mTFP
in the chloroplast colocalizes well with the chlorophyll autofluorescence.
In contrast, the
fluorescence of mTFP in the cytosol does not colocalize with chlorophyll
autofluorescence.
Fluorescence signals from secreted mTFP as well as HumcetFv fused with mTFP
were detected in
the cytosolic area in structures that could correspond to the ER/Golgi.

The results in Fig. 3 confirm, for the first time, successful detection of
our scFv fused with mTFP,
demonstrating the production of secreted scFv in *Chlamydomonas*.
Although
both proteins are
secreted, we were able to capture them while in transit through the
secretory pathway, most likely
within the ER and the Golgi apparatus.

### Secretion Signals

Lastly, we addressed our third question regarding the efficiency of
different secretion signals for our
scFvs by directly comparing the secretion signals from carbonic anhydrase
(cCA), arylsulfatase
(ARS2) and gamete lytic enzyme (GLE) in a single Western blot. In most
cases, the cCA secretion
signal is reported to function reliably and ensures efficient protein
secretion. However, ARS2 and
GLE can serve as alternatives if secretion levels with cCA are too low, e.g.
MUT-PETase[[4]](https://2019.igem.org/Team%3ATU_Kaiserslautern/Results).
Should
secretion efficiency be higher with ARS2 or GLE, it would be necessary to
design new Level 2
constructs accordingly.

We selected positive transformants expressing scFv from constructs 9-14
(**see Fig. 1**). Liquid
cultures were grown to a density of 2\*105 cells/ml for seven days and
proteins in the culture medium
were then precipitated with acetone and analyzed by SDS-PAGE and Western
blotting.

![](https://static.igem.wiki/teams/5758/results/figure4.webp)

**Figure 4:** Comparison of cCA, ARS2 & GLE secretion
signals in
CetFv and HumcetFv constructs 9-14 probed with anti-HA antibodies.
The recipient strain UVM4 was included as a negative
control, and L5 served as a positive control.

As shown in **Fig. 4**, HumcetFv expression coupled with the
cCA secretion
signal resulted in markedly
higher secretion levels compared to its ARS2 and GLE counterparts. In
contrast, the CetFv construct
displays slightly higher expression with GLE and occasionally with ARS2.
Nevertheless, we decided
to continue using cCA for CetFv, as the difference is not substantial and
maintaining the same
secretion signal allows for better comparability with HumcetFv.

These data conclude the initial construction and testing of our Cetuximab
scFVs. In summary, we
successfully showed secretion of scFv both by immunoblotting and by
fluorescence microscopy and
identified cCA as the most suitable secretion signal. Having laid this
groundwork, we next aimed to
explore secretion under different parameters such as culture volume or time.
In parallel, we sought
to refine existing protocols to reduce the workload and minimize potential
hazards from vile reagents.

---

## Optimization of Methods

After identifying cCA as the most effective secretion signal for our scFv
constructs, we began
planning the next series of experiments. One of our primary goals was to
improve the trichloroacetic
acid (TCA) protein precipitation protocol, as TCA is highly hazardous and
difficult to handle safely.
In addition, we aimed to investigate the secretion of our scFvs by varying
parameters such as liquid
culture volume and timing of protein harvest.

### TCA vs Acetone Precipitation

Trichloroacetic acid (TCA) is a highly corrosive and hazardous chemical to
work with. While TCA
precipitation is routinely used in the Department of Biotechnology and
Systems Biology, we were
eager to improve the protocol by skipping the highly corrosive TCA-step, and
precipitated with the
less hazardous acetone, therefore reducing hazards, and saving time.

*Chlamydomonas* cultures of HumcetFv were grown at a density of 2\*105 cell/ml
for six days. One
batch was precipitated with acetone, the other with TCA. The ratio of
protein concentration was
adjusted by loading 20 µl of the acetone-precipitated sample onto the SDS
gel and 4.1 µl of the
TCA-precipitated sample (corresponding to a ratio of ~1:4.29). The remaining
steps of SDS-PAGE
and Western blotting were conducted as always.

![](https://static.igem.wiki/teams/5758/results/figure5.webp)

**Figure 5:** Comparison of TCA precipitation and acetone
precipitation. For both precipitation methods, the
*Chlamydomonas* culture HumcetFv, transformed with the construct 10
and UVM4
(negative control) were used. Proteins
in culture media were precipitated with either TCA or acetone. Samples
from the
TCA HumcetFv lane sloshed into the lane of
the negative control (UVM4), thus explaining the signal visible in the
control. L5 served as a positive control.

As shown in **Fig. 5**, both TCA and acetone precipitation of HumcetFv
from
culture medium yielded
comparable signal strengths, indicating that acetone precipitation is as
efficient as TCA precipitation.
Based on these results, we discontinued TCA protein precipitation and used
acetone precipitation
instead as the standard protocol.

### Volume test

Our long-term goal in the iGEM competition was to establish scFv production
in large-scale
bioreactors. If successful, this would allow for mass production of scFvs
without the need for
numerous Erlenmeyer flasks, thereby approaching industry standards. To begin
with, we needed to
analyze the production of our scFvs in different liquid culture volumes to
determine whether protein
yield changes with culture size.

Liquid cultures of strains producing CetFv and HumcetFv, construct 9 and 10, were grown at a
density of 2\*105 cells/ml
for six days in the following volumes of media (in mL): 6, 10, 20, 50, 100,
200, 400 & 1000. After
cultivation, the culture media were precipitated with acetone and analyzed
by SDS-PAGE and
Western blotting.

![](https://static.igem.wiki/teams/5758/results/figure6.webp)

**Figure 6:** Volume test of cultures transformed with
construct 9 and 10. **(A)** Western blot analysis of the volume
test for HumcetFv. (B) Volume test for CetFv. The culture
volumes (6-1000 mL) are indicated above the respective lanes.

As presented in **Fig. 6**, the immunoblot signals of CetFv appear to be
saturated, yet the signal of
the band below appears to reduce in intensity, indicating a diminishing in
scFv accumulation. This
suggests that CetFv might be well suited for scaling up and could serve as a
suitable candidate for
bioreactor experiments. In contrast, the HumcetFv signal decreased with
increasing culture volume,
suggesting lower secretion efficiency in larger cultures. Based on these
initial scale-up results, we
proceeded by planning and operating several types of photobioreactors (see
Outlook).

### Secretion-Time Test

The secretion-time test provides insights into the temporal dynamics of
scFv secretion in
*Chlamydomonas* enabling the secretion time optimization of production
protocols. Understanding
when and how efficiently our two scFv variants accumulate in the culture
medium is essential for
maximizing protein yield and establishing standardized harvesting
procedures.

![](https://static.igem.wiki/teams/5758/results/figure7.webp)

**Figure 7:** Western Blot analysis of culture supernatants
collected daily from day 0 to day 6 post-inoculation at 2 × 10⁵
cells/ml. The left panel shows CetFv, construct 9, secretion with gradual
accumulation
starting from day 2. The right panel
demonstrates HumcetFv, construct 10, secretion with early onset and
sustained high
secretion levels. UVM4 wild-type strain served
as negative control. Molecular mass standards are shown in
kDa.

The Western Blot analysis of CetFv and HumcetFv in **Fig. 7** reveals
distinct
secretion profiles for the
two scFvs. CetFv shows a gradual increase in protein accumulation starting
from day one, with
relatively consistent levels maintained through day 6. In contrast, HumcetFv
demonstrates secretion
beginning on day one, reaching high levels at day 2 that remained stable
throughout the entire week.
Notably, HumcetFv accumulated to much higher levels compared to CetFv,
suggesting superior
production/secretion efficiency or enhanced protein stability in the culture
medium. The early and
sustained secretion of HumcetFv indicates that it can be harvested as early
as day 3 without
compromising yield, potentially reducing production time. The same applies
to CetFv. This time
analysis establishes the foundation for optimized production schedules.

---

## Proof of concept

As we gained experience and confidence in laboratory work, we began to
develop ideas on how to
move forward. Our primary objective was to demonstrate the interaction
between our Cetuximab
scFvs and the EGFR (Epidermal Growth Factor Receptor). In addition, the
Department of Toxicology
offered us the opportunity to test effects of our scFvs on cancer cell
lines. To perform this assay, we
required large amounts of purified protein and therefore scheduled weekly
protein purification
sessions to meet this demand. Furthermore, we anticipated designing scFv
variants in which the
C-terminal protein tags could be removed by a protease, addressing potential
requirements for future
commercial applications.

### HRV Cleavage Site Design

To commercialize our therapeutic scFvs, it is essential that no
additional tags such as SP20, His-
or HA-tags remain on the final scFv. To achieve that, we designed a new B5
part containing a
PreScission protease recognition sequence (human rhinovirus (HRV) type 14),
which enables the
removal of all C-terminal tags. However, due to synthesis problems with the
repetitive SP20 tag, we
were only able to order the B5 part with mNeonGreen (mNG). In order to
obtain the B5 part with the
cleavage recognition site but without mNeonGreen, we planned to remove the
mNG sequence by
PCR.

![](https://static.igem.wiki/teams/5758/results/figure8-5.avif)

**Figure 8: (A)** Amino acid and corresponding DNA sequences
of the
recognition site of the human rhinovirus type 14 3C
protease. **(B)** Planned Level 2 constructs with a spectinomycin
resistance
cassette, the AR promotor, the cCA secretion
signal, either CetFv or HumcetFv as coding sequences, the RPL23 terminator
and the newly designed part with the
PreScission recognition site, SP20 for glycosylation and optimal
secretion, an 8xHis tag for purification, 1xHA tag for
Western blot detection and mNeonGreen for fluorescence
detection.

After the arrival of the HRV part including mNeonGreen, we assembled Level
2 constructs using
Modular Cloning, according to the scheme shown in **Fig. 8**. Following
correct
assembly, we
proceeded with UVM4 *Chlamydomonas* transformation. After two weeks of
growth
on TAP agar
plates, we picked single colonies for screening of transformants.

![](https://static.igem.wiki/teams/5758/results/figure9.webp)

**Figure 9:** Western Blot screening of transformants
producing
scFvs with the HRV-cleavage part with mNeonGreen.
Numbers above the lanes correspond to the individual transformants picked
for analysis. **(A)**Western Blot analysis of
CetFv transformants. **(B)**Western Blot analysis of HumcetFv
transformants.

From Western Blot analysis shown in **Fig. 9**, multiple strong bands
were
detected for both CetFv
and HumcetFv above 70 kDa, which corresponds to the expected molecular mass
of ~56.4 kDa
including glycosylation. These results confirm the successful expression of
both constructs. In
addition, we performed fluorescence microscopy with the CetFv strain,
analogous to our screening
of mTFP fused scFv variants, to assess whether the observed fluorescence
signals correlate with
our Western Blot results.

![](https://static.igem.wiki/teams/5758/results/figure10.webp)

**Figure 10:** Brightfield, mNeonGreen and autofluorescence
imaging
of CetFv-producing strains containing the HRV and
mNeonGreen part using an Olympus BX53 microscope at 1000x magnification.
Transformants shown are the same as
in **Figure 9A**. Chlorophyll autofluorescence was detected at 440 nm,
while
mNeonGreen was visualized at 490 nm. Both
mNeonGreen and chlorophyll autofluorescence channels were merged for
colocalization analysis.

As shown in **Fig. 10**, both Western Blot (**Fig. 9A**) and
fluorescence
microscopy imaging identify the
same positive transformants. However, microscopy imaging detected one
transformant that did not
show a signal in the Western blot, whereas the Western blot revealed
transformants 6 and 9, which
were not detected by fluorescence microscopy. These discrepancies may result
from gene
silencing/activation events.

Simultaneously, we removed the mNeonGreen sequence from the B5 part by
site-directed
mutagenesis. For this purpose, primers were designed to anneal directly
adjacent to the mNG
sequence, thereby excluding it from the PCR product. After amplification,
the PCR product was
purified, and the original plasmid was digested with DpnI to eliminate
template DNA.

![](https://static.igem.wiki/teams/5758/results/figure11.webp)

**Figure 11:** Primer sequences and agarose gel after PCR
**(A)** Forward and reverse primer sequences. The forward primer
anneals to
the His tag coding sequence and the reverse
primer to the SP20 tag coding sequence, thereby removing the mNeonGreen
sequence located between them. For
each primer, two variants were designed to ensure functionality of PCR in
case one fails.
**(B)** Agarose gel electrophoresis showing PCR products. Numbers 69 to
78
indicate the annealing temperature used
during PCR. “PH” refers to Phire Plant polymerase, which was tested as an
alternative to Q5. PH1 corresponds to the
primer combination of reverse short and forward short, while PH4
represents reverse long and forward short. The
expected plasmid size is 2464 bp.

As shown in **Fig. 11**, we successfully amplified DNA at the ~2.5 kb
mark,
matching the size of 2464
bp plasmid lacking the mNG coding sequence. Following Sanger sequencing
confirmation of the
Level 0 B5 part lacking the mNeonGreen coding sequence, we proceeded to
assemble new HRV
constructs and transformed them into *Chlamydomonas* UVM4. However,
because of
time
limitations, we were unable to screen the new HRV transformants.

### Protein Yield Estimation and Concentration

As mentioned before, the Department of Toxicology invited us to test our
scFvs on cancer cell lines.
For this, however, we required sufficient amounts of purified protein.
Therefore, we established a
routine of weekly protein purifications using the strain harboring
construct 16, which produce
and secrete the HumcetFv scFv with a C-terminal 8xHis tag. This process relied on immobilized metal
affinity
chromatography (IMAC), in which the liquid culture supernatant containing our scFv was applied to
a column loaded with Nickel-NTA resin. Histidine residues of the 8xHis tag
specifically bind to the nickel, while
protein lacking the histidine repeats are washed away. Elution of the bound
protein is then achieved
by applying a high concentration of imidazole, which competes with histidine
for nickel binding,
thereby releasing the protein. Purification also opened the opportunity to
calculate protein yield, an
important metric when evaluating the potential of *Chlamydomonas* for
future
commercialization.

Approximately 1-3 L of liquid culture of cells producing HumcetFv, construct
10, were grown
for seven days. After
harvesting, proteins in the culture medium were precipitated with ammonium
sulfate and subjected
to dialysis for buffer exchange. Following one day after dialysis, the
proteins were centrifuged at
25,000 g for 20 min to remove larger particles, to prevent clogging the
column. Each eluate was
mixed with 4xSDS and DTT buffer and then analyzed by an SDS-PAGE along with a
BSA-standard
for yield estimation. SDS-gels were then stained with Coomassie.

![](https://static.igem.wiki/teams/5758/results/results-figure9-1.webp)

**Figure 12:** Coomassie stained gel & Western blot
analysis of
purified HumcetFv, construct 16.
**(A)** SDS-PAGE after Coomassie staining. Shown are Input (Inp), four
elution
fractions and BSA standards (0.5, 0.25,
0.125, 0.0625 µg) used to generate a calibration curve for protein yield
estimation.
**(B)** Western blot analysis of the same purification for quality
control.
After one day of dialysis (D), samples were
centrifuged at 25,000 g for 20 min. Supernatant (Inp) was loaded into the
Nickel-NTA column, while a pellet fraction was
resuspended and sampled. Flow through samples were collected at the
beginning (FT1) and the end of loading (FT2).
Wash samples were collected the same manner as flow through fractions.
After collecting the elution samples, all
samples were precipitated with acetone and analyzed by SDS-PAGE followed
by immunoblotting. Strong signal bands
in E1-E4 confirm successful purification of HumcetFv.

After Coomassie staining, the gel in **Fig. 12A** was analyzed with
ImageJ to
generate a calibration
curve with the BSA standard to calculate protein yield. The highest yield
obtained was ~49 µg/L,
while the average across multiple purifications was ~32 µg/L. These values
are in line with typical
yields obtained with *Chlamydomonas*. Although these yields appear low
compared to industrial
expression systems, it should be noted that UVM4 strain cultures in TAP
medium can only reach
cell densities of approximately 2\*107 cells/ml. This intrinsic limitation is
the reason why we routinely required liters of *Chlamydomonas* culture every
week to provide sufficient protein for toxicology
assays. Elution fractions containing our scFvs were concentrated using
Amicon filters, resulting in
higher protein concentrations suitable for downstream applications.

---

## Pulldown Interaction-Assay

Demonstrating the activity of our
Cetuximab scFvs towards the EGFR was our primary goal. Proving its
interaction would not only validate our scFvs functionality but also
highlight the potential of *Chlamydomonas* as a future contender for
biopharmaceutical protein production. For this, we went for a pulldown
interaction assay inspired by the work of Kiefer et al., 2022 where the
authors proved the interactions between recombinant hACE2 to the SARS-CoV-2
spike protein produced in *Chlamydomonas*. In their experiment, hACE2,
containing a deca-histidine tag, was immobilized on Nickel-NTA beads,
enabling the spike protein to bind hACE2. Since the spike protein had no
direct affinity for the Nickel beads, it remained bound through its
interaction with hACE2 during washing steps.

In our experiment, we
incubated purchased recombinant EGFR harboring a C-terminal histidine tag
with ammonium sulfate concentrated *Chlamydomonas* culture supernatant
containing Cetuximab scFv (without His tag, construct 9 and 10). 66 µl
Nickel-NTA were
equilibrated with PBS before adding the protein mixture. After incubation
under constant inversion, the supernatant was removed and the beads washed
with PBS and 5 mM imidazole. Elution of bound proteins was then conducted
with PBS and 500 mM imidazole, all samples were precipitated with acetone
prior to SDS-PAGE and Western blot analysis.

![](https://static.igem.wiki/teams/5758/results/figure13.webp)

**Figure 13:** Western Blot
analysis of pulldown interaction assay between EGFR and
*Chlamydomonas* scFv.
Recombinant EGFR carrying a C-terminal His tag was incubated with
supernatant containing only SP20 and HA tagged scFv or negative controls.
Samples of Input (Inp), Supernatant after incubation (S), wash (W) and
elution (E) were analyzed. EGFR was detected using anti-His antibodies,
while scFv, Luciferase, and PETase were detected using anti-HA antibodies.
The His-Hum construct served as positive control.
**(A)** CetFv with and
without EGFR **(B)** HumcetFv with and without EGFR **(C)**
Luciferase negative
control with and without EGFR **(D)** PETase negative control with and
without
EGFR

As shown in **Fig.
13**, both CetFv and HumcetFv displayed weak signals in supernatant and
washing fractions, while strong bands for EGFR and the respective scFv were
detected in elution fractions. In control samples without EGFR, no HA signal
was detected in the elution fractions, clearly demonstrating that our scFv
specifically binds to EGFR.

Although Luciferase should not bind EGFR,
a very weak signal was detected for luciferase in the elution fractions.
This effect is most likely caused by unspecific binding of luciferase to the
nickel-NTA matrix, as it was also seen in the EGFR-free control in **Fig.
13**.
The second negative control, PETase, showed as expected no signal in wash
and elution fractions with or without EGFR.

This result provides
proof of the interaction of *Chlamydomonas*-made Cetuximab scFVs with
its
target protein EGFR and marks a major success of our project!

---

## Resazurin-Assay

After confirming the binding capacity of the scFv
to the EGFR in the pulldown assay, we wanted to test if the scFvs can bind
the EGFR of cancer cells in vitro. This assay is designed to evaluate the
cytotoxic effects of various treatments on cell viability using a
resazurin-based metabolic activity indicator. Resazurin, a deep blue
compound, undergoes reduction to pink resorufin in the presence of
metabolically active cells, providing a quantitative measure of cellular
viability. The colorectal cancer cell lines HT29 and SW48 were treated with
the isolated scFv (HumcetFv) and commercial Cetuximab, which serves as a
positive control to enable comparison with an established inhibitor. Cell
culture media and PBS + imidazol function as negative controls to establish
baseline viability, while saponin (10%) and 5-fluorouracil (5-FU) at 7.5 nM
serve as additional positive controls, representing known cytotoxic agents.
This experimental design allows for direct assessment of the isolated scFv's
efficacy against established inhibitors while ensuring proper validation
through appropriate positive and negative controls.

![](https://static.igem.wiki/teams/5758/results/figure14.webp)

**Figure 14:** Resazurin reduction assay schematic.
96-well plate layout for the resazurin assay treatment with
cetuximab, scFv, and positive and negative controls, with incubation
periods
of 24h and 72h. The experimental design anticipated that cells treated
with
the negative controls would maintain high viability (pink), while cells
treated with the positive controls (saponin and 5-FU) would exhibit
significant cytotoxicity (blue). It was hypothesized that both cetuximab
and
the isolated scFvs would have comparable anti-proliferative effects on the
cancer cell lines.

![](https://static.igem.wiki/teams/5758/results/figure15.webp)

**Figure 15:** Resazurin reduction assay results (72h)
for SW48 and HT29 colorectal cancer cell lines.
**(A) SW48 cells:**
The
leftmost blue column (column 1) represents the blank (resazurin without
cells). Negative controls (PBS + Imidazol, Medium) show high viability
(pink). Positive controls demonstrate expected cytotoxicity, 5-FU shows a
partial effect (purple/blue), while saponin shows strong cytotoxicity
(blue). Cetuximab treatments (left columns) display minimal to no
cytotoxic
effect with pink coloration similar to the negative controls. scFv
treatments (right columns) also maintain predominantly pink coloration,
indicating maintained cell viability across all tested concentrations.
**(B)
HT29 cells:** A similar pattern was observed with the negative
controls
(pink). The positive controls validate the assay conditions: 5-FU shows
partial cytotoxicity (purple/blue), saponin demonstrates strong
cytotoxicity
(blue). Both cetuximab and scFv treatments show no cytotoxic effects, with
most wells displaying pink coloration indicating cell viability,
comparable
to negative controls across all concentration tested ranges.

The results of the resazurin
reduction assay indicate that the therapeutic scFvs purified and isolated
from *Chlamydomonas* culture media did not demonstrate the expected
inhibitory
effects on cell viability in the tested cancer cell lines. While the
positive and negative controls functioned as expected, validating the
assay's reliability, neither the isolated scFv nor the commercial Cetuximab
exhibited cytotoxic activity under the tested concentrations. These findings
prevent definitive conclusions regarding the scFv's therapeutic efficacy on
the cancer cell lines, as the lack of activity observed with cetuximab
suggests that the concentrations used may have been insufficient to elicit
measurable cytotoxic responses.

---

## Outlook

### More single chain Fragments

After sufficient testing of the scFv of Cetuximab, we
initiated a new iteration of construct design and testing of newly
engineered scFvs from Matuzumab, Panitumumab and Trastuzumab. The aim was to
evaluate both their production and their interaction with the respective
target receptors, thereby establishing *Chlamydomonas* as a suitable
host for
the production of various scFvs.

Both Matuzumab and Panitumumab also
target EGFR[[5]](https://doi.org/10.1016/j.ccr.2008.02.019)
[[6]](https://doi.org/10.1371/journal.pone.0163366), making them highly suitable candidates for our study.
Trastuzumab was included, due to its well-established role in breast cancer
therapy. However, it targets the HER2 receptor.

For each antibody,
the sequences of the heavy and light variable chains were adopted[[7]](https://opig.stats.ox.ac.uk/webapps/sabdab-sabpred/therasabdab/therasummary/?INN=matuzumab)
[[8]](https://gsrs.ncats.nih.gov/ginas/app/ui/substances/6A901E312A)
[[9]](https://gsrs.ncats.nih.gov/ginas/app/ui/substances/88162617-7817-4caa-bfbe-bdf2c33bf1b0) and
connected by a flexible linker (GGGS3)[[10]](https://doi.org/10.1038/s41598-022-09324-4). Codon usage, intron insertion
and inclusion of BbsI restriction sites were performed in the same manner as
for the Cetuximab scFvs.

The following Level 2 constructs were
designed:

![](https://static.igem.wiki/teams/5758/results/figure-16-2.webp)

**Figure 16:** Designed level 2 constructs for
*Chlamydomonas*
transformation. All constructs contain a spectinomycin resistance
cassette,
as well as the AR promotor. For secretion into the medium, the secretion
signal cCA was added, followed by either Matuzumab, Panitumumab or
Trastuzumab protein sequences. C-terminal tags were incorporated for
glycosylation (SP20), purification (His tag), Western blot detection (HA)
and cleavage site for the PreScission protease. Lastly, all constructs are
terminated by the RPL23 terminator.

However, due to time constraints, we could not test the
interaction with their respective receptor. This is something we look
forward to addressing in the future. Nonetheless, we can confirm production
of Trastuzumab single-chain fragments derived from *Chlamydomonas*, as
shown
in **Fig. 16.**

![](https://static.igem.wiki/teams/5758/results/figure17.webp)

**Figure 17:** Western Blot analysis of Trastuzumab
scFv-producing transformants. Lane numbers correspond to the individual
colonies tested. UVM4 Nit was used as a negative control, and L5 served as
a
positive control.

### Scale Up

In our efforts to produce enough scFv so that *Chlamydomonas* could
be used as a viable alternative platform to produce therapeutic antibodies,
we conducted research into bioreactors as systems for the growth and harvest
of therapeutic proteins. One such system is a 10 L fermenter shown in
**Fig.
18**, previously used for the fermentation of filamentous fungi. This
bioreactor had not previously been known to be capable of growing
*Chlamydomonas*, especially strains such as UVM4 that we used, which
lacks a
cell wall and is therefore more susceptible to cell damage. The bioreactor
showed potential in growing *Chlamydomonas* to stationary phase in
tests with
the wild-type strain. Unfortunately, constant contamination that occurred
during inoculation of the bioreactor, coupled with technical difficulties
with the automatic water-cooling system, prevented further
experimentation.

![](https://static.igem.wiki/teams/5758/results/figure18-1.webp)

**Figure 18:** 10 liters fermenter

The initial positive results of the
10L bioreactor prompted us to go for more sophisticated bioreactor systems
such as the BIOSTREAM 2 – Benchtop flatpanel bioreactor for 1.8 L. This
specialized bioreactor was our first planned approach into using a
bioreactor designed to be used with *Chlamydomonas*. This bioreactor
features
several advantages specifically tailored for photosynthetic microorganisms,
including optimized light distribution through its flat-panel design,
precise pH and dissolved oxygen control, and gentle mixing that minimizes
shear stress on fragile cell wall-deficient strains like UVM4.

![](https://static.igem.wiki/teams/5758/results/figure19-1.webp)

**Figure 19:** BIOSTREAM 2 – Benchtop flat-panel bioreactor
1.8 L assembly

The BIOSTREAM 2 system addresses many
of the limitations encountered with the conventional 10 L fermenter. Its
transparent flat-panel design ensures uniform light penetration throughout
the culture media, critical for maintaining photosynthetic activity and
optimal growth conditions. Additionally, the system's sterile sampling ports
and automated monitoring capabilities significantly reduce contamination
risks that plagued our previous experiments. We allowed the cultures to grow
for six days before beginning sample collection on days 7-9 to test whether
scFv accumulation occurs in the 1.8L bioreactor system. Both TAP
(Tris-Acetate-Phosphate) and HMP (High Magnesium Phosphate) media were
evaluated to assess scFv production under different nutritional conditions
at this larger scale. This sampling strategy was designed to capture the
accumulation phase when sufficient biomass and protein expression would be
expected based on our previous small-scale experiments.

![](https://static.igem.wiki/teams/5758/results/figure20.webp)

**Figure 20:** Western Blot analysis of supernatants of
cultures grown in the
BIOSTREAM 2 –
Benchtop flatpanel bioreactor. The strain used harbored construct 10 shown
in
**Fig. 1**, producing HumcetFv. Both TAP and HMP bioreactors were run
for six
days before the start of sample collection from day seven to nine for
protein precipitation. For each day, three technical samples were loaded
into the SDS gel. UVM4 served as the negative control, while L5 served as
the positive control.

Western blot analysis of culture supernatants revealed successful scFv
production in both media conditions, with cells grown in TAP medium showing
consistently higher scFv production compared to cells grown in HMP medium
across all time points (**Fig. 20**). These results could be due to the
slower
growth rate in the minimal medium HMP coupled with the bigger cell size when
*Chlamydomonas* grows in HMP. The results demonstrate that the
specialized
bioreactor system can maintain stable protein production while scaling up
culture volumes, bringing us closer to achieving therapeutically relevant
quantities of scFv antibodies from *Chlamydomonas* expression
systems.

In our final weeks of the iGEM competition, we had the opportunity to make one
last attempt to increase cell density and protein yield by using the promising
CellDEG membrane bioreactor. According to the manufacturer, this cultivation
system enables high biomass accumulation by continuously enriching photoautotrophic
cultures bubble-free CO2 supply up to 10%, thereby reducing oxygen stress.
Besides constant CO2 supply, the system also supports high light intensities
of over 1000 µmol photons m-2s-1, ensuring that the entire cultures receive
sufficient illumination.

![](https://static.igem.wiki/teams/5758/results/figurecelldeg3.webp)

**Figure 21:**
Inside view of the CellDEG HDC 4.100 | 9.100 system showing the nine HD100 cultivators used for the cultivation
of CetFv, HumcetFv, and the wild-type cc1960 under continuous CO₂ and high light supply.

We acclimated *Chlamydomonas* cultures of 9-cCA-CetFv-SH, 10-cCA-HumCetFv-SH (**see Fig. 1**)
and the wild-type strain cc1690 (used as a control) in optimized phototrophic production
medium (6xP) for four days [(Freudenberg *et al.* 2021)](https://doi.org/10.1016/j.biortech.2020.124542).
After incubation, eight HD100 Cultivators
were each filled with 100 ml of either strain 9 or 10, while the last cultivator was filled
with the cc1690 strain. All cultures were adjusted to an optical density (OD) of 0.3 at 680, 720, and 750 nm.
Samples from each of the three strains were collected daily for Western Blot analysis and for determination
of cell density for eight days.

![](https://static.igem.wiki/teams/5758/results/results-celldeg2-2.webp)

**Figure 22:**
Growth curve of CetFv, HumcetFv and wild-type (cc1690) strains cultivated in the CellDEG membrane bioreactor.
The orange line indicates the maximum cell density that can be reached under standard conditions in TAP medium.
Cultures were incubated for approximately 187 hours, and samples were collected daily.
The cc1690 culture was diluted after 69 hours of incubation to an OD of 0,3 at 680, 720, and 750 nm.

As shown in **Fig. 22**, despite a slower initial growth phase, the CetFv & HumcetFv strains surpassed the maximum cell density
typically achieved with TAP medium after approximately 80 hours of cultivation.
Following this point, cultures exhibited a rapid increase in biomass, reaching a maximum cell density of 1,44\*108 cells per mL.
After eight days, the cell density obtained in the CellDEG system was almost ten times higher than the maximum density
typically observed under standard TAP cultivation. This increase in biomass raised our expectations for a corresponding improvement
in protein yield.

![](https://static.igem.wiki/teams/5758/results/results-celldeg1-2.webp)

**Figure 23:**
Western Blot analysis of **(A)** CetFv and **(B)** HumcetFv expression in the CellDEG bioreactor.
Samples were collected daily during incubation, precipitated with acetone, and analyzed
by SDS-PAGE followed by immunoblotting. For day 8, additional half and quarter
sample volumes were loaded to visualize signal strength. To compare with standard culture conditions,
Samples previously grown in TAP medium under optimal conditions were loaded after day 4 and 7. The wild-type strain
cc1960 served as a negative control, while L5 sample was used as a positive control.

As shown in **Fig 23**, protein yield is noticeably higher in cultures grown in the CellDEG bioreactor, even
after three days of incubation. This demonstrates that the system is an effective tool for increasing
both biomass and protein production. Although the exact fold increase in protein yield could not be
quantified due to time constraints, these results strongly suggest that the CellDEG bioreactor holds great potential
for future commercialization of therapeutic single-chain fragment production in *Chlamydomonas*.

In addition to developing new methods and systems to increase the yield of
therapeutic scFvs, we also conducted research into patent landscapes and
commercialization pathways for translating our experimental results into a
market-ready product. Our patent research revealed that the
*Chlamydomonas*
strain UVM4 is subject to intellectual property restrictions. We identified
an alternative strain carrying the same gene knockout that enables high
transient expression of recombinant proteins from the *Chlamydomonas*
CLiP
library, designated CLiPUVM. This strain allows continued production of high
amounts of recombinant protein without patent restrictions. As a proof of
concept, we transformed this alternative strain with our cetFv and humcetFv
constructs to assess whether comparable levels of recombinant protein
accumulation could be achieved.

The Western blot analysis
demonstrates successful scFv production in the CLiPUVM strain for both
constructs enabling the production of cetFv and HumcetFv.

![](https://static.igem.wiki/teams/5758/results/figure21.webp)

**Figure 24:**
Western Blot analysis of HumcetFv and CetFv expression in the CLiPUVM
strain
as a patent-free alternative to UVM4. The constructs used were 10 for
HumcetFv and 9 for CetFv (**Fig. 1**) for the production of HumcetFv
and
CetFv.

---

## Conclusions

This leads us to the final conclusion of our iGEM project. In summary, we
were not only able to demonstrate the production, secretion and purification
of pharmaceutical Cetuximab scFvs in *Chlamydomonas*, but also proved
their
functionality, as shown in **Fig. 13**, through interaction with the
epidermal
growth factor receptor (EGFR). Additionally, we conceptualized the use
PreScission protease for cleavage of C-terminal protein tags with
successfully produced scFvs such as Trastuzumab (**Fig. 17**), and
operated
various types of bioreactors for scale up purposes (**Figs. 19,
20**).

We
believe that our results represent noteworthy progress in research and an
important step toward the sustainable and cost-effective production of
biopharmaceuticals using microalgae. While we are very satisfied with the
outcomes, many questions remain unanswered. For example: Does our Cetuximab
scFv have anti-cancer effects resembling those of the original monoclonal
Cetuximab antibody? Do all scFvs coming from *Chlamydomonas* function
effectively? These are just a few of the many questions that need to be
addressed in future studies.

For most of us, this marks the end of
our iGEM journey. We are extremely grateful for the opportunity to engage in
actual scientific research as young and ambitious students. We would also
like to sincerely thank the Department of Toxicology and the Department of
Molecular Biotechnology and Systems Biology for allowing us to work
alongside them and for their continuous support. Finally, we are deeply
grateful to everyone, including sponsors, who supported us throughout the
iGEM project.

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