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.
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.
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).
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.
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.
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.
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.
Now that we obtained the final level M plasmid (pM-156) we can insert it into our host organism : Chlamydomonas reinhardtii.
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.
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).
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).
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 :
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.
A second toxicity range was done with what we thought were more accurate concentrations (figures 15 and 16). The protocol was the same.
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.
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).
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.
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.
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).
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.
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.
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
|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
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.
Sadly, we did not have enough time to repeat these experiments all over again.
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.
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.
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.