IGEM

Lab Work

Experiments Notebooks Proof of Concept Results Safety

Cloning results

Construction level 0 : pL0-294 (BBa_K3836000)

The insert we ordered from Eurofins is cloned into a recipient plasmid pAMG9121 by GoldenGate reaction. To select the plasmids that received the insert, the reaction products were transformed into Escherichia Coli NEB10b bacteria by heat shock and plated on LB plates containing X-gal and the antibiotic spectinomycin. The results can be seen in figure 1, after 24h of incubation at 37°C. A majority of white colonies confirms that most of the recipient plasmids received the insert coding for the antioxidant peptide.

Figure 1: Photos of petri dishes containing bacteria transformed by the GoldenGate product. Left: bacteria transformed by the GoldenGate reaction product with insert. Right: control, bacteria transformed by the GoldenGate reaction product without insert. [Figure description: Photographs of petri dishes. The box on the left contains a large number of white colonies. The box on the right contains more blue colonies]

By observing the two plates, we notice that the control plate has a higher blue/white ratio than the plate with the bacteria transformed with the GoldenGate reaction product. This observation is coherent: the insert is present and the GoldenGate reaction replaces the LacZ sequence with the insert in the recipient plasmid, making the colonie white.

Three white clones and one blue clone were amplified by placing them in liquid culture in LB medium at 37°C for 24h. The plasmid DNA was then extracted and digested with the restriction enzymes BsteII and HindIII in order to confirm the obtention of our plasmid pL0-296. The digestion product is runned on a 1% agarose gel. The profile of the migrated products, visible on figure 2, is confirmed by comparing it to the expected profile for two of our clones.

Figure 2: Electrophoresis of plasmid DNA digestion products by BsteII and HindIII enzymes. Left: expected digestion profile after digestion with HindIII and BsteII. 1st column: Generuler 1kb plus, 2nd column: pL0-294, 3rd column: recipient plasmid, having the LacZ insert. Right: Digestion of our purified plasmids. 1st column: Generuler 1kb plus, 2nd to 4th column: white clones (expected to have received the insert, 5th: blue clone (having). This electrophoresis shows that we have two clones with the correct plasmid (column 2 and 4). [Figure description: The expected migration profile by digestion of the enzymes in question is on the left. This profile varies according to whether the plasmid contains the insert or not. On the right, the digestion profiles of our plasmids identify that 2 clones do have the insert in their plasmid].

We can therefore conclude that we have obtained the level 0 plasmid pL0-294. A second verification was performed by sequencing at Eurofins using a primer binding upstream of the insertion site of our undecapeptide coding sequence (see figure 3).

Figure 3: Sequence alignment between the obtained plasmid and its sequence obtained in silico. [Figure description : The sequence obtained in silico and the one that has been obtained by sequencing are aligned together. The alignment indicates that all the sequence has been inserted in the receiver plasmid]

This screenshot shows that the sequenced plasmid has the same sequence as expected from the in silico model. So we continued by assembling level 1.

Construction level 1 : pL1-226 (BBa_K3836001)

We then assembled our level 1 plasmid: pL1-226. It contains the transcriptional unit allowing the constitutive expression of the undecapeptide. The GoldenGate reaction is performed with the recipient plasmid and the plasmids providing the PSAD (Photosystem I reaction center subunit II) promoter, the PSAD terminator and the undecapeptide CDS. The enzyme used to assemble the level 1 plasmid is BbsI. Cloning products are then used to transform Escherichia Coli NEB10b again. The culture results can be seen in figure 4.

Figure 4: Petri Dishes displaying colonies from bacterial transformation with Golden Gate’s reaction product. This is observed after 24 hours of incubation on Ampicillin and X-gal medium at 37°C. [Figure description : on the left, the insertion of pL1 seemed to have allowed the growth of both blue and white colonies - lots of them. On the right, a few white and blue colonies were spotted on the control condition ]

Again, three white colonies and one blue colony are grown in liquid LB medium. After 24 hours, the plasmids are extracted and characterized by PCR, with primers placed on either side of the transcriptional unit insertion site. Non-recombinant plasmid will carry the LacZ insert. Success of the transformation will be determined thanks to the size of the amplicons : expected amplicons are 717 bp for the plasmid containing LacZ, and 1520 bp if the plasmid has integrated the inserts.

Figure 5: Left = expected size of amplicons with primers GG180 and GG181.Right = Electrophoresis gel. pLacZ is corresponding to the receiver plasmid that has kept the LacZ sequence. Well 1 = GeneRuler 1kb plus DNA ladder. Well 2 = blue colony. Well 3 to 5 = white colonies. [Figure description : On the second well, we can see one band at 700bp, on the third, fourth and fifth ones, there’s also one band, between 1500 and 2000bp.]

By comparing to the expected migration profile, this electrophoresis confirms that we have obtained our level 1 plasmid: pL1-226.

Construction level M : pLM-156 (BBa_K3836003)

The transcriptional unit of pL1-226 as well as the sequence for constitutive expression of the hygromycin resistance gene are integrated into the M-level recipient plasmid by GoldenGate reaction (using the enzyme BbsI). The bacteria were transformed with this reaction product and 24 hours later we again obtained white and blue colonies The transcriptional unit allowing the expression of the AphVII gene (conferring hygromycin resistance) is composed of the synthetic and constitutive promoteur pAR, the AphVII CDS and the terminator of the Rbcs2 gene. The name of this part is BBa_K3836002.

Figure 6 : Pictures of Petri dishes with transformed bacteria with pM-156 GoldenGate product (right) and the control (left). [Figure description : on the left, some blue colonies among a lot of white colonies, on the right, a few white colonies among a lot of blue ones.]

A white and a blue colony are cultured to amplify the plasmids they contain. After pDNA extraction, the plasmids are digested with BbsI (blue colony) and BsaI (white colony). And the digestion product is run on a 1% agarose gel. The digestion profiles show that we have obtained the expected bands. This confirms that we have obtained our M-level plasmid.

Picture 7 : Left = digestion profil expected after digestion with BbsI on the receiver plasmid or BsaI on pM-156. Right = Electrophoresis gel. [Figure description : well 1 = GeneRuler 1kb plus DNA ladder. Well 2 = receiver plasmid (the 600bp band is corresponding to LacZ and the 4600bp to the backbone). Well 3 = pM-156 (the 3175bp band is corresponding to antioxidant insert sequence and the 4600bp band is corresponding to the backbone).]

Now that we obtained the final level M plasmid (pM-156) we can insert it into our host organism : Chlamydomonas reinhardtii.

Transformation results

After the Onishi protocol, UVM4 Chlamydomonas reinhardtii were spread on TAP-agar dishes containing the antibiotic : hygromycin. As you can see on figure 8, no colonies were found on the negative control. Three little colonies, that have grown afterwards (figure 9), were spotted on the positive control, and assess that the transformation was a success. More importantly, 42 colonies were able to grow on hygromycin : among them, some may have inserted the sequence coding for the antioxidant peptide. The colony PCR should answer this question.

Figure 8: Photograph of transformation results of Chlamydomonas reinhardtii. Picture taken on 06/09/21. On the top the negative control with no exogenous DNA, on the left the positive control with Chlamydomonas reinhardtii transformed with plasmid p1-06, on the right cells transformed with plasmid pM-156 carrying the antioxidant peptide sequence. [Figure description : No colonies are visible on the negative and positive controls. A few colonies have grown for the condition where Chlamydomonas reinhardtii was transformed with the pM-156 plasmid carrying de antioxidant peptide sequence.]

Figure 9: Photograph of the positive control. Picture taken on 16/10/21 of the control petri dish containing cells transformed with the plasmid p1-06. [Figure description: Three colonies have grown on the petri dish.]

RT-PCR results

To evaluate the expression of the antioxidant undecapeptide, we performed PCR product run on agarose gel electrophoresis. Several primers were tried to amplify the antioxidant sequence. The first pair of primers we used were only amplifying 14 pb, and no amplification was observed, whereas RT10/RT11 and RT12/RT13 allow the amplification of 19pb and 20pb respectively.

The result for the housekeeping gene CBLP is letting us think that the same amount of genetic material was put into each well (Figure 10).

Figure 10: 1% Agarose gel displaying the amplification of CBLP. Well 1 and 14 : 1kb + gene ruler DNA ladder. Well 2 to 11 : all 10 transformed strains of Chlamydomonas reinhardtii. Well 12 : wild-type non transformed Chlamydomonas reinhardtii. Well 13 : result of reverse transcription without RNA template. [Figure description : No signal in well 13. The signals from well 2 to 12 are equivalent.]

According to figures 11 and 12, we can see that several strains showed a specific amplification of the antioxidant sequence, with more or less expression. Finally, we retain as successfully transformed the strain numbered 2, 5 and 7 (corresponding at wells 3, 6 and 8 in below pictures).

Figure 11: 1,5% agarose gel displaying the amplification of our antioxidant cassette using RT10/RT11 primers. Well 1 and 15 : 1kb + gene ruler DNA ladder. Well 2 to 11 : all 10 transformed strains of Chlamydomonas reinhardtii. Well 12 : wild-type non transformed Chlamydomonas reinhardtii. Well 13 : result of reverse transcription without RNA template. Well 14 : plasmid pM-156 carrying the antioxidant sequence. [Figure description : A band of the same molecular weight is visible for wells 3, 6, 8, 9, 10 and 14.]

Figure 12: 1,5% agarose gel displaying the amplification of our antioxidant cassette using RT12/RT13 primers. Well 1 and 15 : 1kb + gene ruler DNA ladder. Well 2 to 11 : all 10 transformed strains of Chlamydomonas reinhardtii. Well 12 : wild-type non transformed Chlamydomonas reinhardtii. Well 13 : result of reverse transcription without RNA template. Well 14 : plasmid pM-156 carrying the antioxidant sequence. [Figure description : A band of the same molecular weight is visible for wells 3, 6, 8, 9, 10 and 14.]

We could not confirm the presence of the undecapeptide as we do not have specific antibodies that can recognize it. We thought about doing steric exclusion chromatography after protein extraction and try to see, in the low molecular mass products, a band showing up by comparison with a non transformed strain.

Proof-of-concept : In vivo tests results

First, the effects of three antioxidant molecules were evaluated with a concentration range toxicity test (figure 13) : H2O2, Rose Bengal (which you can clearly see in the center of figure 13) and methyl viologen. The aim was to find the correct amount of molecules to kill around 60% of the cells. Each condition was made in triplicate. An equivalent number of wild-type Chlamydomonas reinhardtii were put in each well. The concentrations tested were the following :

Figure 13: Toxicity range of oxidative stress inducing product concentration. 96 wells plate with wild-type Chlamydomonas reinhardtii. The first 3 columns contain H2O2, the fourth, fifth and sixth columns contain rose bengal and the 3 last methyl viologen. [Figure description : Top: Descriptive table of the concentrations of the toxicity ranges. Bottom: The wells are green because of the chlorophyll of Chlamydomonas reinhardtii except those whose concentration of Bengal rose is the highest, where the color is pinkish.]

Cells were cultivated 24h in the presence of oxidant molecules. We collected 50µL of cells and added 10µL of Evans blue, an exclusion dye for which dead cells are permeable to. The observation under an optic microscope allowed us to count alive cells, dead cells and the total number of cells. With all this information, we were able to calculate a survival rate displayed on figure 14.

Figure 14: Table of survival rates of wild-type Chlamydomonas reinhardtii in the presence of different concentrations of oxidant molecules. [Figure description : The survival rate decreases with increasing concentration of toxic compounds.]

A second toxicity range was done with what we thought were more accurate concentrations (figures 15 and 16). The protocol was the same.

Figure 15: Chart of 96 wells plate layout for the second concentration range of oxidants on wild type of C. reinhardtii. [Figure description : For each condition, 3 replicates are performed. The concentrations of of hydrogen peroxide are 0, 5 and 6.5 µM. The concentrations of rose bengal are 0, 6 and 7 µM. The concentrations of Methyl Viologen are 0 and 1 µM. A control on TAP medium is also performed.]

Figure 16: Second chart of survival rates of wild-type Chlamydomonas reinhardtii in the presence of different concentrations of oxidant molecules. [Figure description : The survival rate decreases with increasing concentration of toxic compounds.]

From this point on, we chose to use Rose Bengal as the oxidative-stress inducer, as the results were more consistent.

The next step was to try out the antioxidant effect of the peptide on these non transformed cells, with manganese, when put in the presence of oxidant molecules. An equivalent number C. reinhardtii was put in each well, all conditions were done in triplicate (figure 17). For the antioxidant effect to happen, Mn²⁺, the peptide and pyrophosphate must form a complex, that’s why the experiment is held in a phosphate-enriched medium (TAP+Pi). It was important to review the effect of both decapeptide and undecapeptide, which have the same sequence except for a methionine at the beginning of the undecapeptide.

Figure 17: Chart of 96 wells plate layout for first try out of the protective effect of the peptide. TAP : Tris Acetate Phosphate. [Figure Description : 10 conditions are represented on the table, mimicking the display of the plate. There is the same number of cells in each well. Condition 1 = HSM medium. Condition 2 = phosphate enriched TAP medium. Condition 3 = Rose Bengal and phosphate enriched TAP medium. Condition 4 = Regular TAP medium. Condition 5 = Manganese and phosphate enriched TAP medium. Condition 6 = Manganese, Rose Benagl and phosphate enriched TAP medium. Condition 7 = samed as 3 + decapeptide. Condition 8 = same as 6 + decapeptide. Condition 9 = same as 3 + undecapeptide. Condition 10 = same as 6 + undecapeptide.]

After 24h of incubation, we could not draw any conclusions. First, the concentration of manganese was too high (10mM) and most of the cells were aggregated because of the induced stress. As we learned by doing some research, we realized that manganese, in the concentration with which we wanted to use it, could become a stress for phototrophic organisms. Secondly, in the presence of 6µM of Rose Bengal, all of Chlamydomonas reinhardtii seemed dead. But, more importantly, every condition with the decapeptide or the undecapeptide showed some unusual blue aggregates that none of our Chlamydomonas reinhardtii experts were able to identify and that made cell count impossible (see figure 18).

Figure 18: Pictures taken under the optic microscope after addition of Evans blue evaluating the effect of both decapeptide and undecapeptide. Top left corner : cells in TAP+Pi medium. Top right corner : cells with Mn²⁺ in TAP+Pi medium. Center left : cells with Rose Bengal in TAP+Pi medium. Center right : cells with Rose Bengal and the decapeptide in TAP+Pi medium. Bottom : cells with Rose Bengal, Mn²⁺, undecapeptide in TAP+Pi medium. [Figure Description : Top left corner = a lot of green cells are alive in TAP medium. Top right corner = cells are in fewer numbers and some of them start to agglomerate and are colored in blue. Center left = all cells marked with evans blue, so they’re dead. Center right = wide blue agglomerates that look like cellular debris. Bottom = thinner blue aggregates.]

In order to specify the adequate concentration of manganese to allow our experiment, we then realized a range allowing to establish a toxicity threshold (figure 19). A toxicity range assay was made to quantify the effect of manganese. In each well, we put 200µL of C. reinhardtii liquid culture and different concentrations of MnCl2. Doses of manganese went from 0,25mM up to 10mM.

Figure 19: Chart of 96 wells plate layout for the concentration range of Mn²⁺ on wild type of Chlamydomonas reinhardtii. [Figure Description : in the plate, many manganese concentrations were tested. No Mn²⁺ - 0,25mM - 0,5mM - 0,75mM - 1 mM - 1,5 mM - 2 mM - 2,5 mM - 5 mM - 6,25 mM - 7,5 mM - 10mM.]

Again, cells were incubating 24h in the presence of manganese and Evans blue was used to color dead cells. The proportion of dead cells accumulates with manganese concentration, but starting from 5mM of Mn²⁺, we could witness the development of brown aggregates, that Chlamydomonas reinhardtii is known for when under a lot of metabolic stress (figure 20). So we choose to keep the concentration of manganese at 1mM for the following experiences.

Figure 20: Pictures taken under the optic microscope after addition of Evans blue evaluating the effect of manganese at several concentrations on wild-type Chlamydomonas reinhardtii cells. Top left corner : 0mM. Top right corner : 1mM. Bottom left corner : 5mM. Bottom right corner : 10mM. [Figure description : Top left corner = a lot of green cells are floating. Top right corner = slightly fewer cells are present, but all alive. Bottom left corner = half of the cells are alive, the other half are colored by Evans blue and some little brown aggregates are starting to be formed. Bottom right corner = most of the cells are dead and bigger aggregates can be seen.]

To understand where the blue aggregates were coming from, the effect of the peptides, alone and with Evans blue, were reviewed under the microscope (figure 21).

Figure 21: Pictures taken under the optic microscope. Top left corner : cells with Evans blue in TAP medium. Top right corner : cells with the decapeptide and Evans blue in TAP medium. Bottom left : cells with the undecapeptide and Evans bleu in TAP medium. Bottom right : undecapeptide and Evans blue, no cells. [Figure description : Top left = a lot of green and alive cells. Top right = huge dark blue agglomerates which form a network and are preventing us from seeing any cells. Bottom right and left = both are showing blue aggregates that look like cell debris.]

From this experiment, we were able to conclude that Evans blue and both the decapeptide and undecapeptide were interacting and forming aggregates, preventing us from counting dead or alive cells. At this step, we had to find another way to numerate cells that did not involve Evans blue.

In vivo tests results on transformed cells

The aim of in vivo tests was to see how transformed Chlamydomonas reinhardtii would react in presence of an oxidant, as they should express the undecapeptide. To count cells, we had the idea of adding FDA (fluorescein diacetate), which produces fluorescein when hydrolysed by esterase : FDA only colores living cells.

A range of survival ratios was measured with different concentrations of H2O2 : we have selected the dose of 2,5mM, for a survival rate of 37%. Transformed colonies were incubated 24 hours in presence of Mn²⁺, H2O2 or both. The experiment is again done in triplicate and, as we can see in figure X, we analyzed the mean number of cells of each triplicate. Numbers of living cells are coming from a cell counter after the addition of FDA. The final number of cells were calculated under the form of a percentage in relation to the number of cells without any treatment. After analysis, we retain the strain that showed a greater number of cells in the condition Mn²⁺ & H2O2 than in the condition H2O2 only. The number should also be superior to the Mn2+ & H2O2 control strain.

Figure 22: Result’s chart of toxicity essay on transformed Chlamydomonas reinhardtii cells. The control column is showing the number obtained with the non transformed strain. [Figure description : The first table is giving the triplicate average number of cells counted in each condition (cells only, cells and Mn²⁺, cells and H2O2, cells and both Mn2+ and H2O2). The second table is giving the percentage of cells compared to the condition without treatment. In bold, the number of cells coming from strain 1 and 9 are showing up as the one that may have a resistant phenotype.]

In conclusion, according to this analysis, only strains 1 and 9 could present an antioxidant effect. However, from the PCR outcomes, only strain 2, 5 and 7 expressed the RNA corresponding to the antioxidant peptide.

Nevertheless, multiple biais were found in this experiment. The manganese concentration was still too important and killed 61% of cells on average. We may not have homogenized the cultures enough before taking the sample to the cell counter. But most importantly, the cell counter wasn’t precise enough and we observed some irregularities. Without any cells in the sample, it was identifying about 4,89 * 10⁵ as background noise.

Eventually, we gave more credit to the PCR results than to the in vivo tests to choose which strains to send for irradiation.

In vitro test results

In the first attempt, we tried to characterize the activity of the lactate dehydrogenase (LDH) enzyme after flowing through the amicon tube. The amicon tube was supposed to get rid of H2O2 before putting the substrates (NADH and pyruvate) and reading the absorbance. The activity of LDH is reflected by the decrease in absorbance at 360 nm due to the consumption of NADH during catalysis. The absorbance was too high during the whole experiment so we had to decrease NADH concentration.

In the second attempt, the absorbance was in the wanted range but the enzymatic activity was too low, around 0,0297ΔAbs/min (see figure X). We thought we had lost a great amount of enzymes through the Amicon tube.

Figure 23: Screen capture UV Probe used to calculate the kinetic of enzymatic activity using measurement of absorbance over time, on 08/09/21. [Figure description : the screencapture is showing a decreasing curve corresponding to the decline of absorbance as a function of time.]

In the third try, we characterized the activity of LDH without Amicon tube as we realized the final volume was enough to dilute ROS without disturbing the oxidation of NADH. This time, we wanted to see how the reaction was taking place without any ROS. This experience was repeated three times, results were repeatable and the enzymatic activity was around 0,097 ΔAbs/min.

In the fourth try, we could finally define the effect of H2O2 on the enzyme. Incubation of LDH for 1h with 10mM H2O2 reduces the activity of the enzyme to 17% (figure X).

Conditions	Incubation volumes	Reaction volumes	Average enzyme activity (ΔAbs/min)
LDH only	3,12µL LDH 0,4µM → 1,246pmol 46,88µL Tris-HCl 250mM	338µL pyruvate 16mM → 5,4mM 597µL Tris-HCl 250mM 15µL NADH 12,4mM	0,0995
LDH+H2O2	3,12µL LDH 0,4µM → 1,246pmol 5,1µL H2O2 98mM → 10mM 41,78 Tris-HCl 250mM	338µL pyruvate 16mM 597µL Tris-HCl 250mM 15µL NADH 12,4mM	0,0017

Figure 24: Table of enzymatic activities (Δabs/min) of LDH, corresponding to the mean of triplicates, given by the software UV probe, on 10/09/21. [Figure description : Table describing conditions, incubation and reaction volumes needed to assess the effect of H2O2 on LDH. Results are also noted : the enzyme activity of LDH alone is 0,09995 and the enzyme activity when in the presence of H2O2 is 0,0017.]

On the fifth attempt, we followed the whole protocol to evaluate the protective effect of both decapeptide and undecapeptide on the enzyme, alone or in complexe with Mn²⁺. Unfortunately, no enzyme activity could be detected, even with the enzyme only. We made the assumption that the pyruvate had deteriorated, as it hadn't been made de novo the same day.

Finally, we made a sixth try in which we took the time to review again the effect of oxidation on LDH when it is incubated 1h with H2O2. Results were different this time, there was no difference, in average, of enzymatic activity with or without H2O2.

Figure 25: Table of enzymatic activities given by UV Probe, after 1h incubation. This essay was made on 16/09/21. [Figure description : 5 conditions are listed = LDH only, manganese only, LDH and manganese, LDH and H2O2, and LDH H2O2 and manganese. Enzymatic activities and means are also mentioned : there was no decrease in activity when H2O2 was added.]

Sadly, we did not have enough time to repeat these experiments all over again.

Irradiation results

After an irradiation of 50 or 100Gy, transformed Chlamydomonas reinhardtii returned to our lab. We performed a serial dilution and spread cultures onto TAP-agar plates with the aim of diluting enough to count and compare cell concentration. The results of the cultures under the different conditions are shown in figure X.

Figure 26 : Pictures and charts of the sixteen TAP-agar plates holding the 96 conditions of irradiated Chlamydomonas reinhardtii. Pictures were taken 16/10/21 and irradiation happened on 04/10/21.

Results of this experiment weren’t conclusive. Three of the replicates were uninterpretable, most likely due to an oversight by the experimenter : strain 2 dose 1 triplicate 3 - strain dose 2 triplicate 1 - strain 5 dose 1 triplicate 3. Otherwise, replicates seemed consistent. Unfortunately, non irradiated cells weren’t in greater numbers than the irradiated ones. We couldn’t even tell which of the two doses was the strongest one.

We tried to quantify every condition by counting the number of colonies in the last well, with the biggest dilution factor, to help interpreting (figure X). At dose of irradiation 1, we could say that transformed cells were more resistant to γ-radiations than the control strain, but this conclusion is not true for dose 2.

Figure 27 : table gathering count of Chlamydomonas reinhardtii colonies present in the most diluted wells after irradiation to the Belgian nuclear research center. [Figure description : the first table shows count of irradiated cells in every condition, three of them are aberrant. The second table indicates the averages of previous numbers.]

To conclude, the RT-PCR results showed that the transformation was a success on some of the colonies, but we can’t confirm that the expression of the undecapeptide has a significant impact on resistance to γ-radiations yet. With all that we learned, we wanted to do some more experiments to evaluate the power of the undecapeptide, but time was running out.

Team:Sorbonne U Paris/Results