Team:Valencia Biocampus/Results
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Revision as of 00:44, 13 October 2014
Results
Stability
E. coli is the lab rat of the bacterial world. Not only is E. coli easy to grow, with a doubling time of only 20 minutes, but, genetic modification is fairly easy in strains of these species. Just as well, more details about the molecular biology of this organism are known that any other.
We have performed the most complete characterization of the stability of E. coli in order to confirm its optimality as a chassis for Synthetic Biology. We have subjected wild type strains to a range of stressful conditions (see just below "the limits of E. coli") and measured how our Biobricks behaved in these non-optimal conditions.
1 . The Limits of E.coli
Temperature
E.coli is a fully studied and characterized bacterium. The optimal growth of this specie is 37-38ºC with continuous agitation (200 r.p.m.) but out of this range the growth slows down. First of all, we measured the growth of E.coli at temperatures ranging from 30 to 50ºC. To achieve our goal, we used a fully original technique: instead of an incubator, we used a thermal cycler programmed to display a gradient of temperatures.
The results obtained are shown below:
Conclusion
Is E. coli stable at a wide range of temperatures?
Yes it is! We have detected some variations between the two strains. For example, DH5-alpha resists low temperatures better than XL1-blue, but both grow at a very large range of non-optimal temperatures.
Material fatigue
Our modeling sub-team suggested an original idea. Is there something similar to material fatigue in biology? For example, how would an E.coli culture behave if subjected to a temperature fluctuation? We carried out some experiments with our thermal cycler by cycling the temperature of the medium every minute (37-41ºC) for 3h and maintaining controls at fixed temperatures (typically 37 and 41ºC).
Conclusion
Is E. coli sensitive to fatigue of materials in the form of cycling conditions?
Yes! at least DH5-alpha, but in a positive way. Cycling temperatures resulted in an emergent property: increased growth. In order to check whether this effect was due to a mechanical resuspension (convection) linked to the cycling conditions, we performed a specific experiment with dead, antibiotic-treated E. coli cells (Kanamicine and Cloranfenicol) subjected to extreme (30 to 50ºC) thermal fluctuations. The following figures shows that convection forces might not be the reason of such increased growth. A biological explanation may be hypothesized: cycling conditions allow a range of optimal enzymatic activities to be met.
UV Radiation
UV radiation generally carries out its biological effect by damaging nucleic acids. Depending on the radiation intensity and exposure time the damage caused to the genome is reversible; cells can repair their DNA and continue to grow, although they might carry point mutations. Since UV studies are scarce, our team decided to assay the maximum resistance of E. coli strains to UV radiation of a fixed intensity (340 µW/cm2). The experiment consisted of subjecting spot plated E.coli strains to timed pulses of UV light and comparing the output to a control set of bacteria which received no radiation at all.
We also studied how sensitive these strains are when transformed with our Biobricks. As shown in Fig 5, Biobrick-expressing strains were more sensitive to UV light compared to plasmid-only strains. However, Biobrick7 provided some resistance to radiation in DH5-alpha compared with the controls.
pH & Salinity
Finally, we also characterized E. coli resistance to a range of extreme pH values and salt concentrations. As shown in Table 1, none of the extreme conditions tested resulted in a total inhibition of E. coli’s growth.
Table 1.
pH |
5 |
6 |
7 |
8 |
9 |
XL1-blue |
+ |
++ |
+++ |
++ |
+ |
DH5-alpha |
++ |
+++ |
+++ |
++ |
+ |
Salt |
Without salt |
LB |
LB+1% salt |
LB+2% salt |
LB+4%salt |
XL1-blue |
+++ |
+++ |
++ |
+ |
+ |
DH5-alpha |
+++ |
++ |
+ |
+ |
+ |
BL21. Chromosomal T7 polymerase gene is unstable!
BL21 is an Escherichia coli strain that bears the T7 phage polymerase gene into its genome. Therefore, it is used to express proteins that are controlled by T7 phage promoters, as is the case of three of our Biobricks (Biobricks 8, 9 & 10). However, when we transformed the strain with the Biobricks (blue and yellow chromoproteins), we could not measure any signal. After many tests, we hypothesized that the T7 phage polymerase gene was unstable and did not resist stressful situations such as competence or transformation protocols.
We performed a PCR screening to find out whether the T7 gene was present. We used colonies of BL21 transformed with Biobrick 8 (blue chromoprotein) and grew them on LBA medium. We performed a colony-PCR with a couple of T7 specific oligos designed by ourselves targeting a 1.5 kb region of the T7 polymerase coding sequence. On a 0.8% agarose gel electrophoresis, we saw that the bacteria forming the colonies did not have the gene that codes for the polymerase.
We then decided to check whether our glycerol stock of bacteria contained the gene. Maybe during the process of competence the culture was contaminated. To our surprise, the gene was present in every single colony we analyzed. Therefore, we decided to do a final colony PCR using the bacteria from the glycerol stock as positive controls, and using colonies from the non-transformed competent stock.
Our results reveal that BL21 loses the T7 polymerase gene at some point during the process of making the cells competent, as only three out of the ten colonies analyzed did result in (a rather faint) amplification of the gene.
|
% BL21 colonies with T7 polymerase gene |
Glycerol Stock |
100 |
Competent cells (non transformed) |
30 |
Transformed cells |
0 |
2 . Behavior of different Biobricks in sub-optimal conditions
We show below a summary of our Biobrick characterization program, in which we have subjected selected Biobrick-expressing strains to a range of environmental stresses. Our results clearly show that whereas UV light, pH variation and salt content do not significantly affect Biobrick expression, sub-optimal temperatures can in some cases decrease the Biobrick output. Anaerobic, vacuum culture resulted in a dramatic decrease of Biobrick 2 (RFP) activity.
Temperature Stress
37ºC and 200 rpm are the standard growth conditions of E.coli. Varying these conditions and measuring the growth after three hours, (O.D.600) as well as Biobrick expression (fluorescence), was used to test the effect on Biobrick expression (all data are the average of at least three measurements; fluorescence values are normalized to OD600).
Temperature Fatigue
As described in the first parts of this section “The limits of E. coli”, the strain DH5α grows better under temperature cycling conditions "thermal fatigue" compared to the optimal growth temperature (37ºC), but the same effect was not observed for the strain XL1 Blue. Hence, we decided to check what is the effect of Biobrick expression in XL1 (Fig 6), which reveals a lack of effect of the cycling temperatures on the Biobrick output.
pH & Salinity
Large proteins, including enzymes, are affected by pH. Usually, the catalytic properties of the enzymes are lost and metabolism is halted, which eventually leads to death. The pH range determined for E.coli optimal growth is 6 to 7, but does Biobrick expression affect this? Displayed below are the results of the experiment in which the pH of the medium was varied. The results show that, as expected Bb1 displays a higher fluorescence and that it is stably active independently of the pH value.
DH5α pH
Regarding salinity, a major environmental factor, we also tested the stability of our Biobricks by measuring their output in increasingly salty media (Fig. 8). Again, there was no significant differences associated with salt concentration.
DH5α salinity
UV Radiation
In the section "Limits of E.coli" we tested the resistance of E.coli (Wild Type and transformed strains) to UV pulses, but what happens to the Biobrick when subjected to UV? Does it still work after the UV shock? Below we display the results of Biobrick behavior upon irradiation by measuring fluorescence of the surviving cells. We found out that the fluorescence was not affected by UV exposure.
GFP
RFP
Vacuum
We designed a simple experiment which allowed testing the growth of a facultative anaerobe, like E.coli, not only without oxygen, but under Mars-like vacuum. It consisted of incubating the Petri dishes in deep vacuum during 48 hours. After this incubation period, cells were recollected and O.D.600 as well as fluorescence, determined. Surprisingly, the Biobrick 2 in both strains proved extremely sensitive to vacuum, since normalized fluorescence dramatically decreased compared to the normal pressure-incubated cultures.
RFP
Standardization
Biobrick parts are supposed to be standard, this is, they are expected to behave in a similar and predictable way in different host organisms or “chassis”. In this part of the project we used six different E. coli strains commonly used in molecular biology labs to test whether the behavior of our Biobrick parts was homogeneous among them. Surprisingly, only one of the constructions we tested displayed a relatively standard output among the different strains.
General protocol
- Strike the desired strains on LB plates with the appropriate antibiotic. Allow to grow by incubating at 37°C at least 24 hours.
- Gather a large amount of bacteria with an inoculation loop and transfer to 1 mL of LB. Incubate during 20 minutes at room temperature.
- Inoculate 100 µL of bacterial suspension in a tube containing 3 mL of LB with antibiotic. Perform three biological replica of each culture. The expression of Bb4 was induced with a heat shock at 42 °C for 2 min. The expression of Bb5 and Bb6 was induced by adding doxycycline and IPTG, respectively, to the medium.
- Incubate until OD600 is between 0.1-0.3.
- Measure OD600 or OD595.
- In the case of Biobrick parts 4-6, sonicate an aliquot of the culture, eliminate cell debris, add X-Gal, incubate 6 min at RT, and stop the reaction by adding Na2CO3.
- Measure either GFP fluorescence (Bb1 and Bb3; exc.= 493nm; emis.=505nm), RFP fluorescence (Bb2; exc.= 576 nm; emis.= 592 nm), or absorbance at 630 nm (Bb4, Bb5, and Bb6). Fluorescence measurements were performed in a Hitachi F7000 plate reader, whereas a spectrophotometer was used for absorbance measurements. With Excel, normalize fluorescence by cell density, and calculate the average and standard deviation taking into account the three biological replicas. Represent the data.
Results
The behavior of all the Biobrick parts we tested is shown in Figure 1. From our results, it is evident that the expression of all but one (Bb5) of the constructions clearly differed depending on the host strain of choice. Even more, each strain proved to be more “efficient” at hosting a different Biobrick part. For instance, Bb1 and Bb5 are highly expressed in strain JM109, whereas the expression of the other biobricks was low or nearly undetectable. Note that Bb3, containing the weak promoter J23110, could not be detected in four out of the six strains.
Orthogonality
In Synthetic Biology, two constructions can be considered orthogonal when they only interact at specific and predictable interphases, and do not disturb each other. We have studied this desirable feature of Biobrick parts by combining two constructions in the same cell and comparing this output with the one produced by simple transformations. According to our experiments, that were carried out in two different E. coli strains, Biobrick parts do not behave orthogonal when present in the same cell, being the expression of one of the parts 2 to 3 times higher than the other one. Then, we wanted to go un step further: what is the effect of a simple transformation (a plasmid with a Biobrick part) into the cell architecture? To get some insights about this, we are currently waiting for a proteomic analysis in which the whole proteome of an E. coli strain transformed with a Biobrick part will be compared to that of the non-transformed, control strain.
General protocol
- Strike the desired strains on LB plates with the appropriate antibiotic. Allow to grow by incubating at 37°C at least 24 hours.
- Gather a large amount of bacteria with an inoculation loop and transfer to 1 mL of LB. Incubate during 20 minutes at room temperature.
- Inoculate 100 µL of bacterial suspension in a tube containing 3 mL of LB with antibiotic. Perform three biological replicas of each combination of Biobrick parts.
- Incubate until OD600 is between 0.1-0.3.
- Measure OD600.
- Measure GFP fluorescence (exc.= 493nm; emis.=505nm) and RFP fluorescence (exc.= 576 nm; emis.= 592 nm) in a FP6200 spectrofluorimeter (Jasco, Easton, MD) fluorometer using standard plastic cuvettes.
- With excel, normalize fluorescence by cell density, calculate the average and standard deviation taking into account the three biological replicas, and represent the data.
Results
We performed experiments with two different combinations of Biobrick parts containing a fluorescent protein under the control of a promoter sequence from Anderson’s promoter collection: Bb1 (GFP with strong promoter J23104) + Bb2 (RFP with strong promoter J23104) and Bb2 + Bb3 (GFP with less strong promoter J23110). In all cases, several controls were used: wild type (non-transformed) cells, simple transformations with either the biobrick or an empty plasmid, and co-transformations of the biobricks and the empty plasmids. In all cases there was a high background emission of red fluorescence by the cells. Our Results are shown in Figures 1 and 2.
When in co-transformation, Bb1 and Bb2 showed a clearly asymmetric expression . Bb1 was highly expressed in comparison to Bb2 (2-fold in strain XL1-Blue and 3-fold in DH5α). A similar phenomenon was observed when either Bb1 or Bb2 were cotransformed with empty plasmids. Again, less fluorescence was detected in comparison to the simple transformants. Surprisingly, this fluorescence decay was strikingly different between both E. coli strains: Bb1 strongly lowered its expression in DH5α, whereas Bb2 showed a higher decay in XL1-Blue cells.
However, the result was completely different when Bb2 and Bb3 were present in the same cell. In the XL1-Blue strain, the fluorescence intensity of Bb2 was as high as that of the single transformant, whereas the fluorescence of Bb3 –containing a less strong promoter- dropped dramatically. Fluorescence intensity of Bb3 was not detectable in strain DH5α under our experimental conditions.
With these results with co-transformants, we have to conclude that Biobrick parts, even if controlled by the same promoter and cloned into the same plasmid, are not expressed at similar rates. But, is this lack of orthogonality stable? In other words, is the asymmetric output always the same or it fluctuates depending on the culture? We designed a second experiment to answer this question. By performing several subcultures of the co-transformant strains -with five independent replica of each- and measuring the fluorescence output of each replicate after the subculturing steps, we will check whether fluorescence asymmetry is stable. On the other hand, we have only studied the average fluorescence in a population of cells in our experiments… but what are the variations between single cells? A simple experiment using flow cytometry can help us solve this question…
Open License (and Responsible Research and Innovation)
Not unlike scientific research, RRI is about common sense, about combining self-profit with others’ profit, about taking care of our planet. We believe that being responsible is not the opposite of being selfish; responsibility is just a broad-minded way of selfishness that points at the long-term benefit. And there is no long-term benefit without a sustainable society in a sustainable environment. What is good for our neighbors and for our home must be good for us as well.
Our Proposal: Responsible Research and Innovation as a tool to choose within the IP ecosystem.
Since we, our Human Practices sub-team, are a biotechnology and a law student, we are also glad to see that another key concept in IP is a beautiful science-law metaphor: Jane Calvert’s “diverse ecology” scenario. This image fits well with our interdisciplinary spirit and we found it is very useful because it evokes very well the range of different solutions out there to choose from in order to use a scientific invention in a given way.
Having reached this point, it is obvious which is the next step: there is a whole ecosystem of IP figures and one has to be chosen. How? Our bet is simple: by using RRI as the guiding principle of the decision process.
The following figure illustrates what we propose:
In our view, the decision process has to fall upon the responsibility of the experts or examiners, as it is the case today. But we propose that a formation in RRI for patent examiners and other IP actors is introduced. In the same way that scientist need a basic law background for transferring their knowledge into applied solutions, we envisage a future in which everybody involved in the production of a given IP figure (a patent, for example, from scientists and engineers to ethics specialists, lawyers and other experts) should share a common interdisciplinary formation on RRI. A common language is the first step towards a common goal.
Additionally, we propose in this report two tools that might contribute to avoid misunderstandings and to make a simplified decision process available for a wide audience. In order to accomplish the former objective, we have prepared the two common-languages short dictionaries listed in Annex I. To achieve the latter, we propose, for the first time for the best of our knowledge, a math-based equation that could contribute to make IP issues available to a wide audience (Annex II). We have prepared the equation in two steps. First, we combined the patentability requirements (novelty, inventive step, industrial application) in a mathematically logical way to reach our draft equation (F0). Then, we submitted the formula to several experts to have their feedback, modify F0 accordingly to yield F1 (this reflexive process, combining design with improvement rounds, elegantly evokes a biotechnology strategy called directed evolution).
The very idea of using maths to decide whether something is patentable may seem odd. The opinion of the law experts was useful; but we wanted to have a broader feedback from other social actors. We thus carried out a survey to probe the opinion of iGEMers, scientists/engineers and law students, but also of ordinary people to compare their view on our proposal. The C section of Annex II describes the results of this survey that can be summarize as follows: the majority of the respondents found that a math-based approach could be a useful tool, but should not substitute humans as the final decision makers. We could not agree more on that.
[This is a short extract of our whole Report, under construction]
Annex 1. Brief dictionary of IP and SB Annex 2. Patentability index
The seat of the ST2OOL
One of the goals of our project was to select standard, stable, and orthogonal parts naturally occurring in nature. To do that, we carried out a functional metagenomics strategy aiming at selecting promoters from a library of metagenomic DNA. As a first step, we wanted to isolate metagenomic DNA from environmental samples, but instead of using traditional manual kits, we decided to build a robot able to automatically extract metagenomic DNA for us… It is a pleasure for our team to introduce you our TOOL robot:
“The TOOL” robot
How does it work?
The main goal of this machine was the automation of the DNA isolation process. This robot can be used for both E. coli (mini/maxipreps) and for DNA extraction from metagenomic samples.
The robotic system was developed in a continuous philosophy and all the components were assembled on a piece of wood. In short, it consisted of the following modules:
- The control and powering system: the system was controlled by an Arduino Mega 2560 microchip, powered by a computer via USB. The other components of the machine were powered by an ATX power supply.
- The injection system, which consisted of several screw stepper motors controlled by an A4988 driver. We chose this kind of motors because they allowed a quite accurate control of the position and the speed of the injections. A torque of 35N*m was also included. The system had three-way stopcocks in order to refill the syringes easily.
- The temperature modules: high temperatures were achieved with a system that combined hot water and Peltier cells, whereas low temperatures were achieved by ice cooling. Temperature was measured with a dbs1820 temperature sensor.
- The mixing: a homogeneous mixture of all the reagents was obtained thanks to the use of tubes allowing a turbulent regime.
The complete process was as follows:
- Manually inject a suspension of the cells into 3 mL of ice-cold solution I.
- From this step until step 8, the procedure is automatic (indicated in italics).
- 200 µL of solution II are injected and mixed during the flow into the silicone tubes.
- The mixture is moved through the silicone tubes to a water bath at 65 ºC and incubated for 30 minutes.
- 60 µL of ice-cold Potassium Acetate 3M (pH 5.0) are injected and mixed during the flow into the silicone tubes.
- The mixture is moved through the silicone tubes to an ice bath and incubated 20 minutes at -20ºC.
- The resulting suspension is then filtered through a membrane and insoluble particles are removed.
- An equal volume of isopropanol is injected and mixed with to the filtered solution during the flow into the silicone tubes, and the mixture is incubated at room temperature for 5 minutes.
- At this point, the solution is collected in Eppendorf tubes and these tubes are centrifuged at maximum speed for 10 minutes.
- The supernatant is discarded and the pellet washed with 500 µL of 70% (v/v) ethanol.
- The tubes are centrifuged again at maximum speed for 3 minutes and the supernatant is discarded.
- Finally, the pellet is dried and resuspended in 50 µL of water or elution buffer.
Results
As a proof of concept, we used a suspension of soil bacteria (obtained after several mild centrifugations of a suspension of compost in PBS sterile buffer) to isolate metagenomic DNA. Up to now, we have been able to perform steps 1-8 with our robot, and we are still working hard on the implementation of the final steps of the protocol. Figure 1A shows the Nanodrop analysis on the metagenomic DNA we obtained from the suspension. We isolated pure (A260/A280 and A260/A230 ratios close to 2) and quite concentrated (90.5 ng/µL) metagenomic DNA, the integrity of which was also checked on a 0.8% agarose gel (Figure 1B). Interestingly, we also observed the bands corresponding to the 16S and 23S ribosomal RNA on the gel.
Small budget, big ideas
Table 1 shows a list of all the components and materials used to build the robot. We decided to buy cheap and easy-to-find materials, so anyone can re-build or improve the robot! We only spent around 500€:
MATERIAL | NUMBER | Price (€) |
ARDUINO MEGA 2560 | 1 | 12 |
WIRES | 100 | 10 |
PROTOBOARD | 1 | 3 |
DRIVER A4988 | 5 | 15 |
SCREW STEPPER MOTOR | 4 | 130 |
SYRINGE | 4 | 1 |
SCREW S | 50 | 5 |
ALUMINUM BARS | 1 | 3 |
WOOD TABLE | 3 | 3 |
CLAMP | 8 | 3 |
DRAWER GUIDE | 4 | 3 |
SILICONE TUBES | 5 | 1 |
PERISTALTIC PUMP | 2 | 10 |
BEAKERS | 3 | 7 |
SAW | 12 | |
FISH | 1 | 2 |
DRILL | 1 | 20 |
STOPCOCK 3 WAYS | 6 | 12 |
SCROLL SAW | 1 | 8 |
FILTER | 1 per sample | - |
FILTER HOLDER (own design) | 1 | 4 |
AID SILICONE TUBES | 4 | 5 |
RESISTOR (400 h) | 4 | 3 |
CAPACITOR(0.5F) | 1 | 2 |
LIMIT SWITCH | 2 | 3 |
WELDER | 1 | 15 |
TIN COIL | 1 | 11 |
RULE | 1 | 3 |
LUB | 1 | 6 |
SILICONE PISTOL | 1 | 6 |
SILICONE | 4 | 1 |
SCREW DRIVER | 2 | 5 |
ATX POWER SUPPLY | 1 | 10 |
dbs1820 SENSOR | 2 | 6 |
PELTIER CELL | 3 | 12 |
RELAY | 1 | 6 |
PERISTALTIC PUMP | 2 | 16 |
TRANSISTOR | 2 | 1 |
Hours of work… | A lot! | A lot! |
Selecting natural standard promoters
Once we had the metagenomic DNA extraction performed by our robot, we wanted to clone a library of fragments coming from this DNA in the pAcGFP1-1 plasmid (Clontech Laboratories, Inc.). This plasmid contains a GFP protein placed downstream the MCS, so if a promoter sequence is inserted in the polylinker, the expression of the fluorescent protein is triggered and thus fluorescent transformants can be easily detected.
We digested both the plasmid and the metagenomic DNA with the restriction enzymes EcoRI and BamHI and then purified the resulting fragments (Figure 2).
We set up a ligation reaction, incubated it overnight, and then transformed E. coli DH5α competent cells. We obtained around 300 colonies, but none was fluorescent… We performed a colony PCR with oligonucleotides flanking the MCS of the pAcGFP1-1 plasmid in order to check whether the transformant colonies were carrying inserts of metagenomic DNA, and confirmed their presence as you can see in Figure 3.
We are working now on improving the efficiency of the transformation, so we can increase the probability of "catching" a good promoter sequence… more results coming soon!