Team:Sao Carlos-Brazil/Results
Project Results
Preliminary experiments: melanin protection efficiency
Innoculum
100μL of commercial Saccharomyces cerevisiae previously cultivated in 2mL of YM medium. It takes 48h to get to the stationary phase.
Cultivation
Cultivation at 30ºC in a shaker. The stationary phase is detected when the optic density of a 10-1 dilution is 1,4.
Pre-desiccation inoculum
Petri dishes with solid YM medium were divided into four sectors. In each of them, three drops of 10 μL were added with different dilutions from the initial medium (post cultivation inoculum): 10-5, 10-4, 10-3 e 10-2. The plate was then maintained at 30ºC for the cultivation step.
Desiccation
Five Teflon strips were separated. Each has been selected for a time of irradiation: 0, 1, 5, 10 and 15 minutes. On each strip, nine drops of 10 μL of yeast were added, with three sample types: control, melanin 1 and melanin 2. Each of these types was in triplicate, totalizing 45 drops for Teflon desiccation. Each control drop had 10 μL of medium with yeast. The melanin 1 was prepared with 10 μL of medium with yeast added and desiccated. After desiccation, 10 μL of medium with melanin (extracted from Exophiala sp. medium) was added on top of each desiccated drop and then desiccating it again. The melanin 2 was prepared with 500 μL of medium with yeast mixed with 500 μL of medium with melanin, and each drop of these types was obtained with 10 μL of the resulting medium.
Thereby, a total of 45 drops were desiccated on Teflon inside the laminar flow cabinet. In order to manipulate the samples on the next day, they were maintained in a desiccator with silica gel overnight.
Irradiation
On the next day, samples were irradiated in a chamber with ultraviolet C light, in the presence of a radiometer to quantify the photonic energy that hit the samples. Each Teflon strip stayed in the chamber for a determined time. One strip was not irradiated, only to test cell resistance to desiccation. The other four strips were irradiated for 1, 5, 10 and 15 minutes, respectively. The photonic energy measured were respectively: 525 J/cm², 2849 J/cm², 6140 J/cm² and 9407 J/cm².
Inoculum on Petri dishes
Immediately after irradiation, the desiccated drops were resuspended and plated. Resuspension was carried out with 10 μL of NaCl aqueous solution 0,9% and immediately diluted -1 in a total of 100 μL of solution. To the strip with irradiation time equal to 0, 3 plates were prepared. Each of them divided into six sectors. Triplicates of each type were plated on each plate. In the first, 10-4 and 10-3 dilutions were plated for the three control samples.
Dilutions were always performed with 450 μL aqueous solution with 0.9% NaCl and 50 μL from the previous dilution. The -1 dilution was prepared with 100 μL of the solution as previously described. Thus, in each of the six sectors of the plate, three drops of 10 μL of the respective dilutions were dripped. Example: on the control plate with 0 min of irradiation, two sectors were used to drip three drops on each, both 10-4 and 10-3 dilutions of each triplicate sample.
For time 0, three plates were prepared: control, melanin 1 and melanin 2. The same was done for other times, but with different dilutions. For time 1, the dilutions used were 10-3 and 10-2. For times 5, 10 and 15, dilutions 10-2 and 10-1 were plated. After this procedure, 15 plates with 45 different samples were obtained, and in each one there were three drops (total of 135 drops). The plates were grown overnight at 30ºC.
Colony forming units counting
The following day, the colony-forming units in each plate were counted, always at just one of the dilutions. In general, control samples formed more colony-forming units than melanin 1 and melanin 2 types, which suggests some toxicity of the molecule to yeast cells. Melanin 2 type still had slightly longer survival than melanin 1 in general, but quantitative data have not yet been accounted for (will be as soon as possible). Conclusion: The results obtained by the experiment are inconclusive, where both control and types melanin 1 and melanin 2 survived both desiccation and irradiation. There was no significant cell death after irradiation. The difference in cell death was also not significant concerning the different irradiation times, suggesting that the cell concentration was too high for the experiment, with possible formation of a protective cell layer that ensured the survival of more internalized cells in the sample. The experiment should, therefore, have been replicated with lower concentrations of desiccated and irradiated cells, but due to time constraints, we focused on the stratospheric flight.
Gene Fragments Adapters Removal
All gene fragments that were generously provided by Twist Bioscience had upstream and downstream adapters of 21-22 base pairs. In order to join two or more gene fragments, we needed to remove these adapters. We solved this issue by constructing primers with annealing sites at the fragments’ desired locations. We then conducted PCR reactions with Phusion DNA Polymerase to amplify only the intended gene fragment sequence, therefore removing the adapters. More details about the primers and the PCR reactions in Protocols.
Almost all reactions had some minor undesired amplicons displaying weak gel bands, so we needed to purify the right amplicon (strong band) from the agarose gel. The ones that did not produce such amplicons were directly purified from the PCR reaction.
High Altitude Flight
Initially, we had intentions of conducting two stratospheric flights with our samples aboard Zenith Aerospace’s probes. The first would be a proof of concept carrying non-genetically modified yeasts and the second would actually carry Astroshield to the upper atmosphere. The stratosphere is a perfect place to simulate Martian conditions, given that parameters such as ionizing radiation, humidity, temperature, and pressure are incredibly similar to those found on the surface of the Red Planet.
All these factors combined may generate a different and yet more robust result than simple UVC irradiation tests. Due to technical and mainly legislative issues regarding the exposure of GMOs to the stratospheric environment (more details about this on our Human Practices page), we performed a single flight with three different strains of Saccharomyces cerevisiae desiccated with cuttlefish ink’s melanin. The goal was to understand whether melanin could protect the cells against the stratosphere’s very strong ionizing radiation (mainly UVC).
The idea to conduct the first flight and the methods of sample preparation were based on the work of our instructor, Dr. Ana Carolina, who studies melanin as a way of microbiological resistance against ultraviolet radiation. Three strains were part of the high altitude experiment: S288C, PE-2, and a haploid strain derived from PE-2 (FT2751L). Cells were desiccated with a variety of concentrations and always using the same melanin concentration, plus samples with no melanin for reference.
Aside from our samples, the probe designed, built and operated by Zenith Aerospace carried other experiments, such as one with cyanobacteria and many from middle and high school students.
The probe was launched on September 28 at 11:40 am UTC-3 from USP Campus 2 in São Carlos, Brazil, right after the Brazilian Air Force authorized Zenith to use the local airspace. It was lifted by a Hwoyee 1600 g latex balloon filled with helium.
The balloon burst 94 minutes after launch and the probe landed in a rural area of Tambaú, 78.7 km from the launch site and not very far from the predicted site. The total flight time was 135 minutes. Unfortunately, a problem occurred with the avionics’ SD card and most of the data was lost. However, it is estimated that the probe reached an altitude of 30 – 32.5 km. Other parameters can be inferred based on previous flights.
Zenith’s high altitude probes registered a humidity level below 1%, the pressure was at 0.012 atm and temperature at 14 – 19°C at an altitude of 31 km. Note that the stratosphere is considerably warmer in the upper layer than in the lower layer, where temperatures can drop below -70°C. Radiation levels are substantially higher in the upper stratosphere than in the troposphere, especially considering ultraviolet radiation (mainly UVC).
Flight Results
Yeast survival was measured by CFU counting on drop plates. Two treatments (desiccation with and without melanin) were conducted among three different conditions plus a control group (which was not desiccated): ground desiccated samples kept in desiccator, non-exposed desiccated flight samples (susceptible to all stratospheric variables except radiation) and exposed desiccated flight samples (susceptible to all stratospheric variables, including radiation). All treatments were carried out in triplicate.
Out of all strains, S288C presented conclusive and positive results to our proof of concept. The following image illustrates that yeasts desiccated with melanin survived at a rate significantly higher than yeasts without melanin.
After CFU counting of all 9 drops (3 drops for every desiccated replicate), the average number of CFUs was obtained for every treatment, followed by an adjustment for dilution. Note that in order to resuspend the desiccated spot it is necessary to automatically dilute it 10 fold. The number obtained by this process (N) was then divided by N0, which is the control counting. The following table displays the results of these countings for strain S288C (note that dilutions used to adjust the average are not shown).
|
|
||||||||
|
Replicate |
Control |
Desiccated |
Non-Exposed |
Exposed |
||||
|
Treatment |
No Treatment |
Melanin |
No Treatment |
Melanin |
No Treatment |
Melanin |
No Treatment |
Melanin |
|
I |
15 |
N/A |
167 |
189 |
31 |
38 |
0 |
65 |
|
II |
19 |
N/A |
177 |
158 |
32 |
30 |
1 |
70 |
|
III |
26 |
N/A |
164 |
166 |
33 |
22 |
0 |
63 |
|
I |
19 |
N/A |
181 |
172 |
30 |
77 |
0 |
58 |
|
II |
18 |
N/A |
138 |
139 |
33 |
65 |
2 |
69 |
|
III |
15 |
N/A |
119 |
130 |
26 |
62 |
4 |
57 |
|
I |
N/A |
N/A |
119 |
101 |
25 |
76 |
3 |
55 |
|
II |
N/A |
N/A |
102 |
120 |
29 |
65 |
2 |
57 |
|
III |
N/A |
N/A |
119 |
102 |
35 |
66 |
0 |
54 |
|
Average |
1,8667E+01 |
N/A |
1,4289E+02 |
1,4189E+02 |
3,0444E+01 |
5,5667E+01 |
1,3333E+00 |
6,0889E+01 |
|
Dilution Adj |
1,8667E+06 |
N/A |
1,4289E+04 |
1,4189E+04 |
3,0444E+03 |
5,5667E+03 |
1,3333E+01 |
6,0889E+02 |
|
N/N0 |
1,0000E+00 |
N/A |
7,6548E-03 |
7,6012E-03 |
1,6310E-03 |
2,9821E-03 |
7,1429E-06 |
3,2619E-04 |
The following bar graphics show the results obtained from the CFU counting and post counting data treatment, with survival fraction being N/N0.
To confirm whether our data obtained from the flight was statistically significant, we conducted a Two-Way ANOVA test. The graphics above show all significant comparisons after the analysis. The most important comparison for this experiment was between the no melanin and melanin treatments in the exposed group, of which only the strain S288C demonstrated statistically significant data.
We concluded from this high altitude experiment that melanin indeed protects the yeast cells against stratospheric radiation. This helped the team choose this strain as chassis for follow-up transformations and validate the overall concept of the project.
Results AGA2 Joining
The problemWhen we were trying to join two fragments that together compose the AGA2-linker-4D construct with both fusion PCR and Gibson Assembly, we encountered a major problem. Our forward primer had an undesired amplicon in the middle of one of the fragment. The expected amplicon should be 2909 base pairs and the gel bands were all around 1200 bp (as shown in the image below). The best way to deal with it would be to order a new synthesis but we didn’t have neither time nor the resources to do it.
Marker, amplicon (~1200bp)The solution
To solve the problem without a new DNA synthesis, we needed to remove the primer’s homology to the middle of the fragment. We designed a three step approach to tackle the issue. We first amplified each gBlock individually, as illustrated in the image below.
Marker, Aga2a (1187bp), Aga2b (1722bp)Both required agarose gel purification. The fragment on the left (1187 bp) of the gel displayed two strong bands, probably because the reverse primer location was repetitive and had off-target activity. The beginning of the second fragment (with a size of 1722 bp) was also repetitive, which might explain with the band isn’t as prominent as expected. The second step was to amplify the first fragment using a tailed forward primer. The results are displayed in the following image.
Marker, Aga2a (~1200bp), nucA fusion PCR (2114bp), delURA (~740bp)The third and final step was to conduct a nested fusion PCR. We used a forward primer that only anneals to the previous primer’s tail, which is composed of a sequence that is different from the one that induced the undesired amplicon. Our first attempt at performing it was unsuccessful. We intended to move forward with other attempts but due to time constraints, were unable to get a conclusive result.
nucA S. marcescens
Initially planned to be inserted in yeast alongside many other components that are part of our kill switch, we decided to attempt an individual characterization of this part in E. coli. Although we could not express this part in bacterial cells, some milestones were achieved.
CDS Joining
This parts CDS was divided between two gene fragments provided by Twist Bioscience. So the first step was to join it. We achieved this by using fusion PCR and agarose gel DNA purification. The fragments that its CDS was inserted were both part of our overall kill switch, having 1290 and 916 base pairs, with a homology of 92 base pairs between them. This means the fused amplicon should be 2114 base pairs long. A band this size was visible after carrying out the fusion PCR, as shown below in the highlighted sector.
CDS isolation and amplification
With both fragments successfully joined, the next step was to remove the desired CDS from the whole fragment. In order to do it, we designed a pair of primers with tails consisting of restriction enzymes’ restriction site. The forward primer had a BamHI site and the reverse primer had a XhoI restriction site. The primers worked flawlessly, removing the 801 nucleotide CDS from the fused fragment (demonstrated in the image below).
Marker, fused nucA to CDS (801bp)Taq Incubation
In order to try a ligation in pGEM T Easy vector, the amplicon was incubated at 72°C for one hour to add a hangA with Taq polymerase.
Ligation attempt in pGEM T Easy
During the next few days, we tried to ligate the CDS in pGEM T Easy vector, but we were unsuccessful.
Digestion with BamHI and XhoI
Simultaneously with our ligation attempt in pGEM T Easy, we directly digested the amplicon and the expression vector pET-28a with BamHI and XhoI. We had confirmation that the plasmid was linearized, but couldn’t confirm whether both enzymes cut it or only one. Furthermore, we could not verify if the double digestion worked for the amplicon. The image below shows the digested pET-28a and the supposedly digested amplicon (from left to right: marker, undigested pET-28a, digested pET-28a and digested amplicon).
Marker, undigested pET-28a (~4000bp), digested pET-28a (~5400bp), digested amplicon (~800)Ligation attempt
During the next few days, we tried a variety of ligation protocols for both pGEM T Easy and pET-28a plasmids, but unfortunately we didn’t obtain positive colonies.
URA3 deletion
In order to select the positive candidates of transformations, our project was designed based on the auxotrophic marker URA3. We first constructed a cassette deletion (BBa_K3273000) to knockout the gene in our yeasts. This BioBrick was successfully amplified by PCR (as illustrated below) and then purified.
Marker, delURA (~740bp)The selective medium commonly used to select positive transformation candidates is composed of drop-out amino acids and 5-Fluorooctic acid (consult protocols for more information). Our initial transformation attempts were conducted without 5-FOA because the team couldn’t afford to order it and could not also find any research group that made 5-FOA available to us. We used common YM solid medium to plate the transformation reaction. This was a huge problem due to the fact that no selection at all was in place. So we transferred a few dozen colonies per dish to another YM medium Petri dish and after that, to 96 well plates with Sc URA- liquid medium in order to confirm the phenotype after the knockout, after a suggestion from our sponsor Fermentec. If the colony didn’t grow in Sc URA-, it meant it is possible for the knockout. The following image shows some candidates being cultivated in uracil deficient medium.
Sc URA- medium cultivation of candidates using a 96 well plate. This picture only shows negative candidates. After much work and no positive results, Prof. Cleslei Zanelli of UNESP Araraquara kindly donated 0.5 g of 5-FOA to our team. Our following transformation attempts occurred using the selective medium to plate the reactions.
Candidates were then streaked on another 5-FOA Petri dish, as illustrated below.
After a candidate had grown in the streaking process, an isolated colony was transferred to liquid YPD medium and incubated with rotation at 30°C.
After growth, the candidate was once more streaked on selective medium. This time, the medium was Sc URA-. It was additionally transferred to a microtube containing liquid Sc URA-. Also, the candidate’s DNA was purified and used as a template for a confirmation PCR.
Despite phenotypic tests generating promising results, genotypic tests demonstrated that the amplicons produced by the PCR reactions are the same as the controls. The expected amplicon for our specific primer pair in a control sample is approximately 1600 bp and 740 bp in a transformed sample. We could only obtain 1600 bp amplicons in every tested candidate. The image below illustrates the confirmation PCR results of some of the candidates.
Since we have conflicting genotypic and phenotypic results, some hypotheses may explain the issue. The first one is that there is cross-contamination on the 5-FOA Petri dishes. Yeasts have great plasticity towards new environmental stresses and this may result in false positives, given that we might have selected candidates that were actually adapted non-transformants. Furthermore, when transferring the streaked colony to liquid YM or YPD medium, some contaminants might be transferred along and they might have a higher growth rate than the transformed cells. So when trying to confirm the cassette chromosomal insertion via PCR, the reaction will be favored towards the most abundant DNA template, which in this case would be the contaminants. Point mutations in the URA3 gene might occur such as to make a colony URA3 deficient due to a dysfunctional CDS. However, it is unlikely that this is the case for all tested candidates (more than a hundred in total). The second hypothesis is that confirmation PCR reactions are not working as expected, given that every phenotypic test indicates positive transformants but candidates’ amplicons are the same as the control’s. The designed primers might have had off-target activity even though they were checked on NCBI’s Primer BLAST tool. The third hypothesis is that the deletion cassette (BBa_K3273000) is creating an undesired structure, such as concatenation, therefore creating a sequence that is twice the size of the cassette. This might occur through the yeast’s homology repair mechanism.
To verify the third hypothesis, we digested both the candidate`s and the control`s amplicon with NocI. We chose this enzyme because the URA3 gene has a NocI restriction site and our deletion cassette did not. After digestion, both amplicons were effectively cut, confirming that the URA3 gene was still present in the candidate`s chromosome. The following image shows amplicons after NocI digestion. Control is on the left. The original amplicon was around 1600 bp long, and the digestion produced the expected products.
Marker, control (~1250bp and ~500bp), candidate A (~1250bp and ~500bp)We can, therefore, conclude that we would continue our experiments that include following transformations due to the fact that the desired phenotype was obtained. We would not be as certain of the progress we would make because of our genotypic results, but experimentations would go on.