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Results

Part 1 : Construction of model S17.1 - pCM184 - 5' -3' inserts
The principle here is to provide a plasmid construct to the wild type strain of C. metallidurans CH34, which will allow, in the presence of a recombinase already expressed in it, to perform a homologous recombination of a targeted gene, with a sequence provided in this construct. This sequence then includes two homology sequences of 300 bp each, in 5' and 3' borders of the target gene, as well as the Kanamycin resistance gene in the centre, in order to select the bacteria in which the deletion will have taken place. The strain of E. Coli S17.1, was used and transformed with the construction since it has the ability to conjugate with C. metallidurans, in order to transfer the plasmid it contains to it, without the need for a helper plasmid. The plasmid pCM184, chosen here as the basic vector for the plasmid construction, contains an OriT sequence, allowing the plasmid to be transferred from S17.1 to C. Metallidurans, and the gene expressing oriT recognising protein. It also contains the Kanamycin resistance gene, with an MCS upstream and downstream of it. It is then sufficient to add the two homology inserts 5' upstream and 3' downstream of kanamycin on this plasmid. The four targeted genes are pbrR, pbrT, czcA and czcD. (They have function for metal resistance) Thus, sequences present in the 5' and 3' borders of each of these genes were selected in order to amplify them by PCR on the genome of C. metallidurans. These PCR products were then digested by restriction enzymes, in the same way as the plasmid pCM184, in order to insert the 3' inserts into it by ligation, first, then the 5' inserts in a second step.
An agarose gel was used to summarize all the plasmid constructions obtained (Final Gel 1). This one, makes it possible to prove the presence of the 5' and 3' inserts of 300pb each, by the size of the strip corresponding to the construction in its linear form. Indeed, since the plasmid alone has a size of 6760 bp, it is possible to differentiate the plasmid containing only the 3' insert (size 7022 bp), or containing both 5' and 3' inserts, in its linear form (7299 bp). Similarly, these vectors were digested by the two restriction enzymes used during their construction, in order to highlight the presence of each of the inserts. Due to the malfunction of the ligation step for some constructs, only the pbrR gene was targeted for deletion in C. metallidurans. Thus, the conjugation was performed between strain S17.1 - pCM164 - inserts pbrR 5'-3' and C. metallidurans CH34 wild type. These two strains were then placed in direct contact on a solid medium and incubated for 5 days at 30°C. After conjugation, the colonies of C. metallidurans transformed and selected by the presence of Kanamycin, were isolated in order to recultivate them and perform PCR on the genome, in order to prove the absence of the pbrR gene (size 438 bp), replaced by the Kanamycin resistance gene (size 816 bp). Amplification was therefore carried out from primers hybridizing to the two homology sequences 5' and 3', in order to obtain a size of 1416 bp on gel if the homologous recombination took place, or a size of 1038 bp if it did not take place. The gel results for the pbrR gene then showed that the construction of the C. metallidurans strain ΔpbrR was successful.
Part 2 : Construction of model S17.1 - BioBrick+Pan
To enable ethanol production to be provided mainly from glucose, present in sludge or produced by the degradation of cellulose, a fusion protein adhB - pdc is provided to C. metallidurans, to enable the conversion of pyruvate into ethanol. The aim is then to provide glucose by digesting the cellulose present in the sludge, in order to provide an additional source of carbon to produce ethanol, without impacting the growth of bacteria. For this purpose, the BBa_K1122673 share was used to optimize this conversion of pyruvate to ethanol. The part was digested by the restriction enzymes EcoRI and SpeI, in order to insert it into the plasmid pBBR1MCS-2. This plasmid was used because of its replication capacity in both E. Coli and C. metallidurans, by the presence of the replication origin oriV. Similarly, this plasmid contains an MCS in the vicinity of the Lac promoter, allowing induction of the expression of a gene provided by IPTG.
SDS Gel 1 : Expression test of the fusion protein adhB - pdc from BBa_K1122673 part (noted BB) by IPTG induction in E. Coli S17.1 and under the Pan promoter control (noted Pan) in C, metallidurans CH34 on SDS page gel
Thus, this construction pBBR1MCS-2 - fusion was introduced by transformation into S17.1, in order to perform a first expression test of this fusion protein by induction with IPTG (SDS gel 1). This induction test, has been migrated to SDS - Page gel, in order to highlight the expression of the fusion protein. From the sequence of this fusion protein, it is possible to theoretically calculate the molecular weight of this protein, which is then 100.92 kDa. On the gel, a band appears and intensifies with induction time at about 100 kDa, corresponding well to the expected results. Then, a strong constitutive promoter in E. Coli and C. metallidurans (Pan promoter) was added to the construction, so as to express at least as strongly the fusion protein as with the Lac promoter in S17.1 and constitutively. To do this, the promoter and the plasmid construct were digested by the restriction enzymes BamHI and EcoRI, so that the promoter was inserted by ligation upstream of the fusion protein gene. This new construction pBBR1MCS-2 - Pan - fusion, is then introduced in S17.1, to first test the expression of the fusion protein and compare it to induction by IPTG.
SDS Gel 2 : Expression test of the fusion protein adhB - pdc from BBa_K1122673 part (noted BB) by IPTG induction in E. Coli S17.1 and under the Pan promoter control (noted Pan) in C. metallidurans CH34 or on C. Metallidurans with pbrR gene deletion (noted ΔpbrR) in different culture conditions and with or without activation on 284 medium supplemented by lead and cadmium durring 15 minutes (noted activated), on SDS page gel
The results on SDS - Page gel show an expression approximately equal to that of induction by IPTG after 20 hours of incubation. (SDS Gel 2) These results show that this promoter allows the strong and constitutive expression of the fusion protein in E. Coli S17.1. This plasmid construction was then introduced into C. metallidurans by electroporation. All the steps in the implementation of this pBBR1MCS-2 construction containing the Pan promoter, followed by the fusion protein, were demonstrated by electrophoresis. (Final Gel 2)
Similarly, the expression of the fusion protein, under the control of the Pan promoter in C. Metallidurans, was shown by SDS - Page gel and compared to the induction expressions in IPTG or under the control of the Pan promoter, in E. Coli S17.1. gel (SDS 1 and 2) The results of the SDS - Page gel thus show a similar expression in C. Metallidurans, and in S17.1 (induction with IPTG or under the control of the Pan promoter). This then proves that the fusion protein is well expressed in C. Metallidurans and in a strong and constitutive way.
Part 3 : Proof of concept
Cellulose
Before performing the sludge field tests, we performed various tests under medium conditions 284 (heavy metal supplemented media) in which we added different sources of sugars to create minimum media in order to demonstrate that our bacteria can consume glucose. We prepared a first medium 284+Glucose as a control and a medium 284+paper to reproduce a glucose source similar to sludge. The paper is first digested by a mix of cellulase from Trichoderma reesei. Glucose consumption capacity will first be assessed by measuring the OD of the cells growth.
OD measurment during time of bacteria in 284 Glucose medium
For medium 284 Glucose (Figure 284 Glucose), we observe an increase in OD, which indicates that bacteria can use it to grow. However, it should be noted that growth appears to be slightly higher for the bacterium Δpb than for the CH34+BB_pan strain. In a second step, the same measurements are made in medium 284 Paper (284 Paper), here again, bacterial growth can be observed.
OD measurment during time of bacteria in 284 Paper medium
The latter is again more important for the bacteria Δpb than for the strain CH34+BB_pan. In parallel, an assessment of the amount of glucose in the medium was made via a DNS assay. In both cultures, there is a decrease in the glucose level in the environment, which shows that it is indeed consumed by the bacteria CH34+BB_pan and by Δpb.
The bioavailable glucose level in medium 284 Glucose (Figure Glucose rate in 284 Glucose medium) is fixed and more important at the base. Glucose consumption also seems similar for the two strains CH34+BB_pan and Δpb. In contrast to the glucose level in condition 284 Paper (Figure Glucose rate in 284 Glucose Paper). Indeed, in the latter, cellulase continues to digest cellulose and there is therefore a part of the consumption that is compensated by this "production" of glucose.
Nevertheless, the high rate of decrease after 24 hours shows a higher sugar consumption in this condition. It was also found that CH34+BBB_pan appears to consume less glucose than Δpb, which corresponds to the slower growth of this modified strain of metallidurans. The large size of the BioBrick and the very strong promoter that precedes it force the bacteria to produce the protein of interest, which consequently leads to a decrease in its division rate and therefore a lower glucose consumption due to the lower number of bacteria. It can therefore be conceptually assumed that the modified bacteria are capable of growing in sludge because they are able to use cellulose from paper digested via cellulase.
Heavy metals
To verify the effectiveness of our bacterial construction on metals, we carried out a first bacterial culture in medium 284 supplements (284 supp) in Lead (0.5mM) and Cadmium (2mM). These concentrations were not taken at random, the lead concentration corresponds to the minimum growth inhibitory value (MIC) of C. Metallidurans CH34 and that of Cadmium is less than half of this MIC and still activates the mechanisms of Cadmium resistance. The 3 bacterial strains tested, E. Coli S17-1, C. Metallidurans CH34, and C. Metallidurans ΔpbrR, were pre-cultured for 36 hours in Broth Medium 3 containing no heavy metals and then cultured for 24 hours in the tested medium (284 supp). An Optical Density follow-up was performed to evaluate the potential inhibition of C. Metallidurans ΔpbrR by Lead. Finally, the different cultures were stopped and centrifuged to recover the bacterial pellet and supernatants separately. The 2 samples were determined for lead and cadmium by ICP, and the pH was measured in the supernatants.
Optical Density Tracking
According to the follow-up of the Optical Density in 284, ΔpbrR and S17-1 saw their growth limited in 21 hours of cultivation. On the other hand, the results show exponential growth from CH34 to 22 hours of cultivation.
pH results
The pH measurement allows visualizing the impact of the bacterial strain on the pH of the medium.
It should be noted that since the solubility limit for lead is 7 when the pH is acid below this value, it becomes more and more soluble. On the other hand, when the value is basic, lead tends to create aggregates.
We can notice that strain S17-1, significantly acidifies its environment thus facilitating the solubilization of lead and thus an increase in its concentration in the supernatant of the media. On the other hand, C. Metallidurans CH34 is alkaline in its media, which must induce aggregation of the lead in the medium and therefore the decrease of the concentration in the supernatant.
Lead Assay at ICP
Lead assay at ICP indicates a lower concentration in the supernatant for bacterial strains S17-1 and CH34, near or lower concentrations than control, indicating for S17-1 solubilization of lead bound to acidity. medium, and an efficient CH34 aggregation mechanism reducing the amount of lead in solution.
On the other hand, ΔpbrR which no longer has a lead efflux mechanism has a richer lead environment.
Here we can find a small amount of lead retained by bacteria or aggregated in S17-1 because the bacterium does not have a strong system of aggregation of lead unlike CH34 and also has an acidic medium.
For the bacterial strain CH34, the amount of lead in the bacterial pellet may very well reflect the aggregation of a pH-related lead coupled with aggregation related to the resistance mechanisms of the bacterium.
Finally, for ΔpbrR, the concentration in the bacterial pellet must certainly be related to the alkaline pH of the medium and therefore to the aggregation of lead, accentuated by the absence of resistance and efflux system of the modified strain.
Cadmium assay at ICP
For cadmium, we can see here the lowest concentration in CH34 indicating an efficient membrane aggregation system.
Here, the results indicate a good aggregation of cadmium in ΔpbrR and CH34, thus an efficient but weaker aggregation mechanism. This slight difference is certainly due to the difference between bacteria CH34 and ΔpbrR.
Part 4 : Construction of the model ΔpbrR - Pan - fusion protein
By the same method as during the construction of the CH34 - Pan - fusion protein strain, the ΔpbrR strain was transformed by electroporation in order to provide it with the expression construction of the fusion protein. Thus, this new strain has been tested for the expression of the fusion protein under different culture conditions. This strain was grown in BM3 medium, in a minimal medium of 284 supplemented with lead and cadmium and presenting only glucose as a carbon source, as well as in medium 284 containing cellulose and in the presence of cellulases in order to degrade it into glucose and other reducing sugars. The expression of the fusion protein is observed after 20 hours of incubation at 30°C, by SDS - Page gel (SDS Gel 3).
It is then possible to see a slightly variable expression between the different conditions. The expected results were a stronger expression in the presence of Cadmium, due to the promoter Pan.(1) Indeed, since bacterial concentrations here are relatively low, these differences in expression could not really be observed.
Part 5 : Application in sludge
Cellulose
After proof of concept, the same tests as before are performed in autoclaved sludge (AB) to test our bacterium under real application conditions. First, OD monitoring is performed in sludge containing cellulase or not containing cellulase (Figure Autoclaved Sewage).
A similar bacterial growth of Δpb+BB_pan bacteria with or without cellulase is observed. This allows us to see that cellulase has no effect on the growth of our bacteria. In a second step, the DNS monitoring made it possible to monitor the consumption of glucose in the sludge autoclaved by the bacteria.
A decrease in the available glucose concentration can be observed under both cellulase and non-cellulase conditions (Figure Glucose rate in Autoclaved Sewage). This corresponds to the results found in Part 3. First, it can be noted that glucose consumption is higher in the cellulase-free condition despite bacterial growth in both conditions. This can be explained on the one hand because the autoclave has released sugars that bacteria can use to develop... but the condition with cellulase allows to degrade more of them, which buffers the loss related to bacterial development. All these experiments have therefore enabled us to demonstrate the survival of our modified C. metallidurans in a glucose-rich environment and the use of this sugar in the metabolism of the bacteria. Our model has also succeeded in operating in real conditions in autoclaved sludge. From the SDS gels pages, we also showed that our bacterium produced the fusion protein through the implementation of BioBrick. So all that remained was to measure the ethanol produced to complete our results... Unfortunately, due to many problems, we were unable to achieve them on time. We ordered a dosage kit in June that never arrived, so we found a plan B using a densitometer (which is usually used to measure the alcohol content of wine). Unfortunately, it was not sensitive enough. Finally, we had the idea to carry out a colorimetric assay using alcohol dehydrogenase, but we were unable to implement it due to a lack of time.
Heavy metals
Followed by Optical Density in the Purification Sludge
According to the monitoring of Optical Density, bacterial growth is performed uniformly in sludge. Determination of pH in sewage sludge
The pH values observed on the cultures in the sludge of purification are all in basic conditions. On the other hand, we can see slight acidification of the medium by S17-1 as in the proof of concept in the medium 284supp. Determination of lead at ICP in sludge
The results here demonstrate a high concentration of lead in the dry matter for ΔpbrR and CH34. This certainly demonstrates a strong aggregation of lead due to the mechanisms or the alkaline pH of the medium.
The concentration values in the supernatants appear much lower than in the dry matter. Nevertheless, a slightly lower concentration can be observed for strain CH34 and a higher concentration for S17-1.
Finally, in the bacterial pellet, we can see a higher concentration of lead attached to the bacteria of the genus CH34 and equivalent results in S17-1 and ΔpbrR. This demonstrates the loss of the lead resistance function in the modified ΔpbrR strain. Determination of Cadmium in sewage sludge
Cadmium concentrations are higher in the dry matter of CH34 and ΔpbrR. These results could be evidence of aggregation by CzcABC systems of its two strains, inducing sedimentation at 300RCF Cadmium, although this remains to be verified in the following results.
Here the solution concentration values in the supernatants are substantially the same for the 3 strains.
Here we can see that cadmium concentrations in bacterial pellets are very low. This indicates that membrane aggregation systems are very inefficient in CH34 and ΔpbrR. We can explain this by the concentration in the sludge of purification too weak to allow the activation of its last ones.
Reference
1. A metal-repressed promoter from gram-positive Bacillus subtilis is highly active and metal-induced in gram-negative Cupriavidus metallidurans. - PubMed - NCBI. https://www.ncbi.nlm.nih.gov/pubmed/20517979.