Notebook | RPTU-Kaiserslautern

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.

**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.

**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.

**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.

**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

**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.

**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.

**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.

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

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

**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).

**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.

**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.

**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.

**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.

**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.

**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.

**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.

**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.

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

**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.

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

**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.

**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.

**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.

**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.

**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.

**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.

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

**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.

**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.

**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.

**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.

**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.

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

**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.

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

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

Choose Week