Difference between revisions of "Team:Newcastle/Notebook/Lab/AlternativeConstructs"

This page documents the wet lab experiments relating to our resistor constructs. We also maintain a separate library of frequently used protocols that are referenced from this page with any amendments.

20/06/16

After the InterLab study, we made streak plates from the colonies we had grown. We regrew all the samples on lysogeny broth (LB) agar with Chloramphenicol (1:1000). We did this to isolate a pure strain of the transformed InterLab E. coli, therefore allowing us to grow up a new, genetically-identical plate. Our lab supervisor, Dr Matthew Peake, showed us the correct streaking technique as the Computer Science students had not learned this technique before.

22/06/16

After analysing the trial InterLab results, we decided to re-plate up the positive control to ensure that we would have enough colonies to carry out the InterLab study again.

28/06/16

Today we made a microbial fuel cell by following the University of Reading's protocol.

We sourced the material such as the neoprene gaskets, carbon fibre electrode material, cation-exchange membrane, J-cloth from Professor Ian Head, Dr Ed Milner, and Dr Paniz Izadi from the School of Civil Engineering and Geosciences, Newcastle University. We also sourced electric wires with crocodile clips and a multimeter from the Electronic and Electrical Engineering Department here at Newcastle University.

First, we prepared the 1 M glucose solution, 0.02 M potassium hexacyanoferrate (III) solution, 10 mM methylene blue solution, these were made up in a 0.1 M potassium phosphate buffer. Please refer to our microbial fuel cell protocol for information on how we did this.

Then, the four Perspex^® components of the fuel cell were then bolted together to make the two compartments of the fuel cell. Neoprene gaskets were provided to prevent leaks from the cell.



Before we start to assemble the fuel cell, we rehydrated the 2.5 g dried Baker’s yeast (Saccharomyces cerevisiae) in 5 ml of the potassium phosphate buffer. Next, 5 ml of the 1 M glucose solution was added to the yeast and mixed well.

We then cut out and folded two carbon fibre electrodes, as seen in Figure 2. One electrode was then inserted into each of the chambers made from the Perspex®.

Two pieces of J-cloth were then cut out and placed into each chamber of the fuel cell, on top of the electrodes. This is to prevent the electrodes from touching the cation exchange membrane.

A neoprene gasket was placed on each half of the fuel cell. The two halves were then placed together with the cation exchange membrane sandwiched between them. The two halves were then tightened by passing four bolts and tightened with the wing nuts. Although we were warned not to over-tighten the nuts as it would distort the cell and allow liquid to weep out. We did find that a lot of our liquid leaked out of the cell and we believe it may be due to the over-tightening of the nuts.

We added 5 ml of 10 mM methylene blue solution to the yeast suspension. After stirring the mixture, we used a clean syringe to add the yeast mixture to one-half of the fuel cell. In the other half of the fuel cell, we syringed around 10 ml of the 0.02 M potassium hexacyanoferrate (III) solution. The multimeter was then connected to the electrode terminals using the wires and crocodile clips.

Our results showed that we had an overall voltage of 397 mV. Although this result was really impressive, it would not be enough to power our light bulb component of the board. Therefore, we shall now work on improving this part and seeing if we can increase the voltage.

Today, we also grew up some liquid cultures for the InterLab study, which we then left to incubate overnight at 37 °C with agitation set to 220 rpm.

29/06/16

The InterLab was carried out on 29^th July. This was a practise run as our Sample 2 had not transformed well. We believe this may have been due to the fact that the competent cells had been carried over from one building to another and not been on dried ice. After a lot of confusion with the protocol, we managed to get the InterLab up and running. It was good to have this practise run as we now know what to do for the final run. For example, we were confused by having to dilute down to an OD₆₀₀ of 0.02, we now know to do this quickly and have a rough idea of what dilution to make.

30/06/16

Liquid cultures were regrown overnight at 37 °C at 220 rpm, until they were required again for the InterLab study.

05/07/16

The InterLab was carried out again. This time, we used the iGEM InterLab protocol exactly, as well as using a new plate reader that our lab had on loan. The ThermoScientific Varioskan Lux Plate Reader had the ability to shake and incubate, so we were able to run for the full six hours without interrupting the cycle. Although this was a good way to test the InterLab study, we wasted a lot of time at the start playing around with the software. This allowed the OD₆₀₀ value to increase from 0.2 by the time we started the cycle. We also had issues with condensation on the plate reader lid, this altered the data towards the end of the experiment as the condensation increased. This meant that the results from this protocol could not be compiled into the InterLab data, as the protocol was not identical to the other iGEM teams.

The other plate reader experiment was done by following the protocol: taking a sample every hour for 6 hours and putting it on ice. Then FI and OD of the samples were measured all at once. The results were mostly consistent with only a few “out of range” replicates. One limitation that might have impacted the results was that even though the samples were diluted to 0.02 OD₆₀₀ using the right calculations, there was no time to check with spectrophotometer if they were diluted to that value in practise. However, we decided to repeat the experiment again due to the protocol being changed.

11/08/16

We PCR'd the pSB1C3 and RFP device to serve as both a test run of PCR operation for later experiments and also to give us a source of pSB1C3 plasmid for later transformations and device assembly. To check our resulting DNA matched the device we used we performed gel electrophoresis on the sample. As the device is 2070 bp in length we expected clear bands around 2000 bp. To perform the gel electrophoresis we used our standard gel protocol with one variation, instead of running the gel for 40 minutes at 90 V we ran the gel for 80 minutes at 90 V as after the first 40 minutes it was not possible to clearly distinguish the bands. Additionally we chose to use 1% gel as in the protocol because the pSB1C3 construct is greater than 2000 bp long (2070 bp). Our result is shown in the below gel image.

As you can see from the image our gel tore when removing the comb. We suspect this is a combination of using a low agarose concentration and that the agarose we used was old. For future experiments, we have noted to use a higher concentration of 1.5%.

More importantly, you can clearly see banding at the 200bp marker which confirms that our sample contains the desired device. There are some artefacts which we think could have been removed through the use of a PCR clean up kit. As we believed we had successfully isolated the plasmid we froze this sample to be transformed later.

12/08/16

We grew E. coli cells in different media to see which ones they survived better in. This was a rough guide before we did the final experiment to see which media we should use in the final thing. For this experiment, we inoculated each of the media types (listed below) with some of the left over InterLab E. coli. The liquid media were then left in a 37 °C at 220 rpm for 24 hours. After the allocated time period, growth was measured by a simple yes/no to whether the media had turned cloudy or not (Figure 3). The data can be seen below in Table 1.

Table 1. Bacterial growth in various media

Growth Medium	Growth
LB (10 ml)	Growth
LB (10 ml) and 0.25 mol of Sodium Chloride	Growth
M9 (10 ml)	Growth
0.5x TBE (10 ml)	No growth
0.25 mol of Sodium Chloride and 20% Glucose Solution	No growth
0.25 mol of Sodium Chloride	No growth

INSERT IMAGE HERE

Figure 3. Bacterial growth in various media

19/08/16 - Electrochemical Impedance Spectroscopy

Electrochemical impedance spectroscopy is an experimental technique for characterizing the dielectric properties of a medium. In our case, the media in question are various concentrations of LB broth, salt solution and gold nanoparticles.

The experiment measures impedence which is the opposition to the flow of alternating current (AC). We have chosen to use AC because the AC resistance is often much higher than the DC resistance, this results in more energy lost through joule heating, which is the process we are trying to exploit. Moreover, an alternating current should not cause electrolysis. This is due to the switching nature of the current. In an AC system, each electrode alternates between being the cathode and anode, spending half the time in each state so any changes that take place should be undone. This assumes identical electrodes, and in reality some electrolysis will take place but the amount will be less than with a DC source.

The value we are interested in is the resistance between the two electrodes, rather than the capacitance across them although both values are collected in this experiment (shown below). Ideally, we would like our system to be purely resistive so it will be interesting to also see the energy storage capacity of the system.

To measure the impedance we model the media as a passive complex system. In this model the system consists of both a resistor ($R_{Ohm}$) to dissipate energy and capacitor ($C$) to store energy. Additionally, some of the energy may leak from the storage ($R$). This is shown in the circuit abstraction of the system below.

The value we want to determine is $R_{Ohm}$, the resistance of the media. This will allows us to determine the power output ($=I^{2}R$) of our cells and therefore the heat dissipated.

To measure the Ohmic resistance of the system we used a potentiostat which maintains a constant voltage across the system and impedance analysis software to compute the resistance. This work was carried out in the Stimming group in the school of chemistry with supervision from Dr. Jochen Friedl.

Set Up

To conduct our experiment we used 3 of our molded microfluidics chambers, made according to our original protocol. One for each medium. Into each chamber, we inserted copper wire electrodes, if identical length and such that they spanned the entire width of the inner chamber. We then filled the chamber with fluid: 0-1 M NaCl solution, 0-100% stock LB and 0-100% 0.1 mM gold nanoparticles in PBS. The electrodes were then hooked up to the potentiostat as shown below.

We then did frequency sweeps to get a Nyquist diagram of the system and determine the ohmic resistance. Between each sweep, the chamber was flushed with distilled water 3 times before adding the new fluid.

Results & Discussion

For our purposes the resistance is the most important value, so only that data will be presented here. If you would like to see all the data you can download a copy of our full results.

The first media we tested was our distilled water, to check that the instruments were producing sane values. This resulted in a resistance of 0.289 MΩ which is a reasonable value to expect. Following this, we tried different concentrations of salt solution in 0.2 M increments up to 1 M solution. As you can see from the data even a small amount of salt drastically reduced the resistance of the water. This is what you would expect since the oxidation of the Cl ions at the anode allows charge to flow.

The best fit for this data series is a power series ($R^{2}=0.9873$). This means that initially small changes in concentration cause a large change in the resistance until the solution becomes saturated and this starts having less of an effect. This is clearly shown on the below graph.

For us, this confirms two important things. Firstly, that we can adjust the conductivity/resistivity of our media easily by varying the amount of salt. For example, increasing the amount of salt in LB. Secondly, that only a small amount of salt is required to improve conductivity. This latter point is important because our bacteria are unable to survive at high salt concentrations due to osmotic pressure. Most E. coli strains can only tolerate up to 0.5 M NaCl.

The second medium we used was LB. We would expect this to follow a similar trend to the NaCl solutions as it is the salt components of LB that allow it to conduct electricity.

Indeed, we do so this trend, albeit with higher resistances due to the further dilution of the salts for 40-100% LB in water. It appears to us that the result for 20% LB is anomalous.

This gives us useful baseline resistances for estimating the best LB concentration to use to generate our desired heating effect. The downside to LB however, is that it is difficult to replicate the preparation each time, compared to a minimal media like M9. We had hoped to also test M9 media as part of this experiment but precipitates had formed in our preparation.

Our final media was gold nanoparticles in PBS. As with the salt solution, we were investigating whether we could finely control the resistance of the media using additives. As pointed out previously, our bacteria are not tolerant to salt and it is metabolically active which means we could be interfering with cellular processes by altering the concentration. This may have an adverse effect on protein production. Gold, by contrast, is not metabolically active so we can add it to the growth media without any effect on the cells.

We had hypothesized that adding the gold nano-particles would decrease the resistivity of the media, that is the gold nanoparticles would facilitate better conduction. The gold nanoparticles that we used are a 20 nm suspension in 0.1 mM PBS from Sigma-Aldrich.

Because the gold is suspended in PBS we used that as the dilution medium. We added gold nano-particles to 0.5x PBS at concentrations of 0-100% (e.g. 20% nanoparticle solution to 80% PBS). The data we collected is shown in the below table.

Our data runs contrary to what we expected, increasing the number of gold nanoparticles increases, the resistance of the solution, as you can see from the graph below.

Unfortunately, this means that we will be unable to use the gold to decrease the resistance, though we can use it to increase the resistance if required. We are more likely to have to decrease the resistance rather than increase it, since the power is proportional to $I^{2}R$ we can improve the power dissipated in the media (and therefore the heating effect) better by increasing the current rather than resistance. This is illustrated in the below graph which shows that the increase we gain from varying the resistance grows linearly whilst the varying the current allows the power to grow exponentially.

These values are linked, $I = \frac{V}{R}$, so there is a trade-off between increasing or decreasing the resistance. Further experiments are required to determine what the optimal resistance for best power output is. However, we now know that using salt and or gold nanoparticles we can vary the resistance in either direction which is a useful tool.

It occurred to us during this experiment that the gold may also affect the heating rate of the medium. We will conduct future experiments in this area to determine this effect.

Computing Required Voltage

We can use the data we have collected from this experiment to determine the voltage required to heat the media in our microfluidics chambers. We do this by linking the energy needed to heat water, given by $E = C_{w}\cdot \Delta T\cdot mass$, and the energy dissipated by a resistor. In the equation for the energy required to heat water $C_{w}$ is the specific heat capacity of water. This formula can be used for any liquid for which we know the specific heat capacity. Thanks to our collaboration with Exeter we know that LB media behaves similarly to water (since water makes up the majority of the media) so we can assume a similar specific heat capacity. We had performed bomb calorimetry experiments to determine the exact specific heat capacity of LB but these were unsuccessful. The specific heat capacity we use here is 4 J/Kg. From our empirical experiments with microfluidcs we found that a heating time of 3 minutes was best to prevent pressure build up from electrolysis. This means the required energy is $E = 4 \cdot 0.1 \cdot (3 \cdot 60) = 72W$ as our chamber holds 1 ml of fluid, weighing 0.1 Kg.

The equation for the energy dissipated is $E = Q \cdot U = I \cdot s \cdot U = \frac{U^{2} \cdot s}{R}$.

By relating the two, $C_{w}\cdot \Delta T\cdot mass = \frac{U^{2} \cdot s}{R}$ and solving with the average resistance we find that to get the desired heating effect in our microfluidics chambers we need a supply voltage of approximately 30 volts. This is the DC voltage, as we intend to use AC this is actually the root mean square value, and the peak voltage required is actually $V_{p} = V_{RMS} \cdot \sqrt{2} = 30 \cdot \sqrt{2} \approx 42V$.

22/08/16 - Replicating Stanford Experiment

Our lightbulb is based on work done by undergraduates at Stanford University as part of their BIOE 44 'Fundamentals for Engineering Biology Lab' course. This work investigated using the E. coli heat shock response to allow the bacteria to respond to electrical current. One of the first experiments we did sought to replicate part of this work to identify the temperature changes that we could induce in LB media.

For our experiment we added 300 ml of LB broth to a standard gel electrophoresis chamber (we chose this value as this is the usual amount of TBE used to cover the gel). We then performed two experiments to heat the medium. The first of these was to keep the current constant at 400 mA, allowing the power supply to vary the voltage to ensure this current as shown below.

As in the original experiment we stopped the power supply at the following time intervals in order to take temperature measurements: 0, 1, 2, 5, 7, 10, 13, 16, 19, 22, 25, 28, 31, 34, 37 and 40 minutes. The temperature was recorded using an alcohol thermometer and the power supply switched back on.

When keeping the current constant we recorded the following data.

This showed us that it is possible to heat LB media using purely electrical effects and that it is possible to get the required $\Delta T$ to induce the heat shock response (10 ℃) as the temperature range in this case was 16 ℃. It was faster to reach our desired temperature difference (=13 mins) than to reach the peak temperature at 37 mins). This is because as the temperature rises the system reaches an equilibrium where heat is lost as fast as it is gained, this is shown by the flattening of the below graph. It is useful to know this as we would like to have our system respond quickly, including time for transcription and translation. This would mean to get our response time down we would have to increase the current or reduce the amount of media.

The original value quoted in the Stanford write-up is a voltage of 65V which they report as a current of 400mA. We suspect that the current is more important for the heating effect since the power dissipated in the media ($P = I^{2}R$) is proportional to the square of the current which is why we ran the experiment both with fixed voltage and fixed current power supplies.

As you can see in the below image, electrolysis of the LB media takes place, as evidenced by the bubbles of hydrogen and chlorine gas forming on the surface of the media.

As electrolysis uses up free ions in the media (mostly chlorine in reduction at the anode) the conductivity is reduced and a higher voltage is needed to produce the same current ($V=IR$). We predict therefore that maintaining a constant voltage will produce less of a heating effect as the current will drop as electrolysis occurs.

We repeated the same experiment with fresh LB (cleaning the chamber with water and then deionized water) but with the power pack set to maintain a constant voltage of 65 V rather than current. The data we collected is given below.

In this case, it took both longer to achieve a temperature difference of 10 ℃, 31 mins, and the overall temperature difference was lower, 10.5 ℃ than when the current was kept constant. As shown in the graph there is a lot less variation in the data, we suspect that this is because it is harder for the power pack to maintain constant current than constant voltage.

This experiment confirmed that maintaining a constant current produces a better heating effect than maintaining a constant voltage. Going forward, we will be using current values to characterize our system rather than voltage values.

The main problems identified by this experiment are; the time taken for the temperature to change 10 ℃ and the amount of chlorine produced in the LB by electrolysis. We would prefer our temperature change time to be around 3 minutes, rather than 13 minutes. To achieve this aim we will be investigating the heating effect on much smaller volumes of LB as this should heat faster. To solve the chlorine problem we will investigate using an AC power source as this should limit the amount of electrolysis that occurs due to the switching nature of the electrodes in this scheme.

19/08/2016 - Gold Nanoparticle Heating Experiment A

We wondered if the gold nanoparticles could be used to enhance the heating effect within the microfluidic chamber, and reduce the time taken for the requisite temperature change to induce the heat shock response.

We found little correlation between the results, and decided not to proceed further with this approach given our time constraints.

22/08/2016 - Rhodamine B Flourescence Experiment A

Following on from our Electrochemical Impedance Spectroscopy work we wanted to determine the heating effect of applying our computed voltage to LB media. Because the heating is being performed in microfluidics chambers it is difficult to measure directly using a thermometer. We found that the thermometer crushes the chamber causing liquid to leak from it when it is not inserted carefully. To overcome this issue we investigated using fluorescent dye as a temperature measure. In particular we chose the well used and characterized dye Rhodamine B (Karstens, T., 1980).

Rhodamine B has the emission spectra shown below with a peak emission at 565nm. It has the property that the fluorescence yield varies with temperature. It is this property which makes it useful for measuring temperature in situations where a thermometer or other technique would be too invasive, such as in biological samples (Chen & Wood, 2009).

Before using the dye in our experiments we first wanted to test that we could measure the fluorescence signal using the equipment we had available. To do this we proceeded as per Shah et al. (2009) which pertains specifically to microfluidics experiments and prepared a solution of 0.1 mmol/L Rhodamine B in LB broth.

We then heated our solution to 55 °C and allowed it to cool. At various temperatures shown in the dataset below, we took a 0.5 ml sample and measured the fluorescence using a plate reader. The data we collected is shown below.

We aborted the experiment because we did not appear to be getting correct data, there was no variation in fluorescence. We believed this could have been because the plate reader was set up incorrectly to measure the emission wavelength of the Rhodamine B and so postponed the remainder of this experiment until we could use the new plate reader.

02/09/16 - First Bulb Current Test

We breifly tested the DNA2.0 lightbulb construct (htpG) in our microfluidics chambers. We placed 1.25 ml of transformed E. coli liquid culture after 24 hours growth into two chambesr using the 'DNA2.0 1(a) 50 31/08/2016' sample.

We observed before we conducted the experiment that the cells were not glowing on exposure to a UV LED as we would expect. We then subjected one chamber to 12 mA current (DC) for 90 seconds and the other to 15 mA for 60 seconds. It was originally intended that we apply the current for longer as we found it takes 2-3 minutes for our desired temperature change in LB at these currents, however the chambers leaked through the PDMS-slide interface which caused the circuit to break early and the power supply to shut off.

To prevent this from happening in the future we think it would be best to adapt our current chamber designs into 3D printed ABS which would form one complete sealed unit so that no leak could occur.

We then recovered the liquid remaining in the chambers and plated them out to see if they grew overnight. Note that due to the leakage the recovering volume was very small (~0.5 ml).

References

Chen, Y.Y. and Wood, A.W., 2009. Application of a temperature dependent fluorescent dye (Rhodamine B) to the measurement of radiofrequency radiationâ induced temperature changes in biological samples. Bioelectromagnetics, 30(7), pp.583-590.

Karstens, T. and Kobs, K., 1980. Rhodamine B and rhodamine 101 as reference substances for fluorescence quantum yield measurements. The journal of physical chemistry, 84(14), pp.1871-1872.b

Shah, J.J., Gaitan, M. and Geist, J., 2009. Generalized temperature measurement equations for rhodamine B dye solution and its application to microfluidics. Analytical chemistry, 81(19), pp.8260-8263.