Difference between revisions of "Team:Uppsala/Proof"

 
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            <h2 class="text">Design</h2>
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<div class="col-lg-8 text">
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            <div style="margin:auto; float: none;" class="col-lg-8">
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<h2> Proof of concept</h2>
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<p>
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As a proof of concept we did 2 rounds of experiments on our heat shock chip, one where we compared its transformation efficiency to that of regular heat shock transformation done off-chip and one where we compared transformation efficiency on the chip at different temperatures.
 +
<h3>Heat Shock Chip</h3>
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<p>
 +
During the summer we made over 30 chips and numerous successful heat shock transformations using only 8.4 ng of DNA and 5.9 μL of competent cells.
 +
</p>
 +
<p>
 +
Bacterial growth was seen on nearly all of the chip transformed plates. In other words, the transformation worked on our chip and it greatly reduced the amount of reagents needed since colony growth was observed on plates were only 6mL of cell/DNA suspension was heat shocked. The number of colonies for each trial was added by counting all of the five plates for each trial as one replicate. That left three replicates for each chip. Calculations of colony forming units per microliter DNA were made and the mean for each chip is shown in Figure 1. We found that the transformation efficiency was much higher in the conventional heat shock than on our chip.
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</p>
 +
<p>
 +
The plates with bacteria transformed on the chip showed many colonies, considering the small amount of cells and DNA that were plated. A large variation in number of colonies between different trials was observed. That is, two transformations carried out on the chip in the same manner could yield a varying number of colonies.
 +
</p>
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<p>
 +
The cleanliness of our chip was good as little to no colonies grew on the plates with only SOB run through the chip nor on the plates with only cells (negative control).
 +
</p>
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<figure>
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<img class="img-responsive img-rounded" src="https://static.igem.org/mediawiki/2016/9/9a/T--Uppsala--Microfluidic_Results_Fig2.png
 +
"/>
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<figcaption>Figure 1. Mean transformation efficiencies of the different chips (1-3) and conventional transformations (0). Transformation efficiencies calculated by: # colonies on plate/g of DNA plated. Error bars show standard deviation, n=3.</figcaption>
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<br>
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<br>
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<br>
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<figure>
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<img class="img-responsive img-rounded" src="https://static.igem.org/mediawiki/2016/e/ed/T--Uppsala--Microfluidic_Results_Fig1.png"/>
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<figcaption>Figure 2. Plates after transformation with chip. Two different trials show the variation in efficiency. Variation could be due to human error.</figcaption>
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                <img class="img-responsive img-rounded" style="margin:auto; clip: rect(0px,500px,0px,0px);" src="https://static.igem.org/mediawiki/2016/2/2a/T--Uppsala--Chip_sketch.png" />
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                        <div class="mybutton text-center results panel-default">
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                            <div class="panel-body"> Raw data </div>
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<div class="col-lg-8 text">
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<div class="row">
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                    </a>
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                <hr>
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                <div class="panel">
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                <h3>Design process</h3>
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                    <div class="col-lg-12 collapse" id="collapsible-2_1">
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                <p>
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<hr>
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                    The microfluidics group were in charge of designing, manufacturing and maintaining the chips that were created. We went over several iterations of designs until we settled on the box designs.
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<h3> Raw data </h3>
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                </p>
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<p>
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        </div>
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Here the raw data of every transformation is presented alongside the comparison transformations we did.
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<div class ="row">
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</p>
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            <div class="media">
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<figure>
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<div class="media-body">
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<figcaption>Table 1.</figcaption>  
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                <p>
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<table class="tableizer-table">
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                    We had 3 main phases of designs.
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<thead><tr class="tableizer-firstrow"><th> Trial. </th><td>3</td><td>4</td><td>5</td><td>6</td><td>7</td><td>8</td><td>9</td><td>10</td><td>11</td></tr></thead><tbody>
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                </p>
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<tr><th>Chip model</th><td colspan="3">1</td><td colspan="3">2</td><td colspan="3">3</td></tr>
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                    <h4>June</h4>
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<tr><th>No. of colonies</th><td>964</td><td>1096</td><td>505</td><td>911</td><td>1074</td><td>55</td><td>85</td><td>771</td><td>1029</td></tr>
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<p> Flat, thin and large designs with many features. During this period we tried to make a mould with multiple features on it. This design paradigm was quickly scrapped mainly due to the moulds (and therefore the chips) easily becoming warped during the curing process. We still gained some valuable insight and were able to stresstest the 3D-printer and find its limits. The chip in Figure 1 is about 7x3 cm (slightly larger than a microscrope slide).
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<tr><th>Transformation efficiency (cfu/ng DNA)</th><td>34.9</td><td>39.6</td><td>18.2</td><td>33</td><td>38.8</td><td>1.99</td><td>3.07</td><td>27.9</td><td>37.2</td></tr>
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                </p>
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</tbody></table>
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</figure>
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<br>
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<br>
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<figure>
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<figcaption>Table 2. </figcaption>
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<table class="tableizer-table">
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<thead><tr class="tableizer-firstrow"><th>DNA concentration(ng/µl) </th><td>47</td><td>47</td><td>47</td><td colspan="3">0.56</td></tr></thead><tbody>
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<tr><th>No. of Colonies</th><td colspan="3">>1000</td><td>170</td><td>72</td><td>64</td></tr>
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<tr><th>Transformation efficiency (cfu/ng DNA)</th><td colspan="3">42.5</td><td>304</td><td>129</td><td>114</td></tr>
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</tbody></table>
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</figure>
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<p>
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According to the results presented in Table 1 and Table 2 the heat shock chip
 +
did not reach higher efficiency than conventional methods, at least with the comparison metrics used. There was also very high variation in the transformation efficiency among the experiments done on on the chip(which were done using similar/identical reagents). The efficiency varied from 1,99 in experiment number 8 up to 39,6 in experiment number 4, which is a 20-fold variation in efficiency. On a per plate basis the highest efficiency we had was
 +
123 000 and the lowest was 0 (8 out of 50 plates had 0 colonies), with the average
 +
transformation efficiency being 36.  
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</p>
 +
<p>
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During the experiments we could identify
 +
several potential causes which could cause this large variation of results. The setup
 +
of the chip involved running heated water from a beaker through the chip and this
 +
means that there can be a chance that there is higher or lower thermal leakage than
 +
expected, leading to the transformation efficiency being affected. Also we were not
 +
sure whether heat shocking at 42 degrees for 35-40 seconds is the optimal way to do it.
 +
Since the heat is conducted much better and faster through the chip from the heating
 +
channels and because the cells are in a much smaller volume of media the cells are
 +
heated more rapidly maybe a lower/higher temperature should be used a shorter/longer
 +
time. Furthermore we noticed that there was a very large variance due to the human
 +
factor(i.e. who was actually conducting the experiment) and that experience in the
 +
procedure played a large role in how successful the transformation was. This was
 +
likely due to the primitive and direct way cells were fed into and out of the chip (using
 +
a syringe to push in/out cells and eye-measuring to see whether cells have entered the
 +
chip makes for inaccurate timing and a lot of back and forth).
 +
</p>
 +
<hr>
 +
</div>
 +
</div>
 +
</div>
 +
<h4>Results for the temperature comparisons</h4>
 +
<p>
 +
For each transformation at a set temperature the amount of formed colonies can be viewed in figure 3. Figure 3 shows the data points for the triplicates at each temperature having a large spread of the amount of formed colonies. Only the transformations at temp. 55 ◦C showed formed colonies for each of the three replicates.
 +
</p>
 +
<figure>
 +
<img class="img-responsive img-rounded" src="https://static.igem.org/mediawiki/2016/8/85/T--Uppsala--Microfluidic_Results_Fig3.png"/>
 +
<figcaption>Figure 3. Number of colonies formed after transformation at each temperature. The crosses represent each replicate, the circle is a contamination check, and the rhombs are the average amount of colonies at each temperature.</figcaption>
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<figure>
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<div class="media-left">
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<a href="#">
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                <img class="media-object img-rounded" height="400" width="250" src="https://static.igem.org/mediawiki/2016/3/35/T--Uppsala--Fig1_Early_Prototye_Our_Designs.png" />
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</a>
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<p>
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</div>
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The highest average value for formed colonies was at 55 ◦C for the heating fluid, giving 30 colonies whilst heat shocking at 65 ◦C and 75 ◦C yielded 21 and 14.7 colonies. The average (avg) number of colonies at each temperature drops slightly when going from 55 ◦C, 65 ◦C to 75 ◦C since the latter two contain replicates with zero number of colonies.
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    <div class="row">
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</p>
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                    <h4>July</h4>
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<p>
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<p>Smaller, thicker rectangles. These chips were designed to be used for a single purpose. Size-wise, these were about 2/3rds of the chip in Fig. 1 and were reinforced below with a grid-like pattern to counteract the warping. However there were still some problems with warping (about one out of four turned out to be good), so we went back to the drawing board to create our finalized design.
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The avg value for the number of colonies at each temperature was used to determine the average transformation efficiency, represented by the cfu/µg value and is presented in Table 3.  
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                </p>
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</p>
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            <h4>Late-July/August</h4>
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<figcaption>Table 3. Average transformation efficiencies at different temperatures.</figcaption>
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<p>The basic design is a box which is 2.2x3.3x2.5 cm. It has a thick base and thick walls which contain the actual workable feature area. The base and walls severely reduce warping, and also allows a higher degree of stability during PDMS pouring and curing. The feature area is where channels can be placed and is the part of the design which will actually be translated into a PDMS chip. The feature area is 1.6x3.0 cm on the finalized designs.
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<table class="tableizer-table">
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        </p>
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<thead><tr class="tableizer-firstrow"><th>Temperature (°C)</th><th>Transformation efficiency (cfu/ng DNA)</th></tr></thead><tbody>
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        <p>
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<tr><td>55</td><td>4.11</td></tr>
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            Early in the summer it was decided that we would primarily work toward creating a functional chip for heat shock, and if there was time to make a electroporation chip and design an incubation feature/chip. Unfortunately there was only time enough to be able to comprehensively test the heat shock chip. The electroporation chip was designed, printed and assembled but there was not enough time to test the transformation capabilities of the design, mainly due to difficulties in getting the electroporation electrodes firmly and correctly attached to the chip.
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<tr><td>65</td><td>2.88</td></tr>
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        </p>
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<tr><td>75</td><td>2.01</td></tr>
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        <h3>Our designs</h3>
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</tbody></table>
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<img class="img-responsive img-rounded" src="https://static.igem.org/mediawiki/2016/8/82/T--Uppsala--design_heatshock_annotated.png"/>
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</figure>
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        <p>
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            A schematic view of the heat shock chip can be seen in Figure 2. The heating channels (in purple) are 850 µm wide and 600 µm high. The heat shock channel itself is 300 µm wide at its thin part and 800 µm wide at the broader parts, it is 500 µm high in all parts. The total running length of the heat shock channel is 2.276 cm and the part of the heating channel which runs parallel with the heat shock channel is 1.41 cm.
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        </p>      
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<img class="img-responsive img-rounded" src="https://static.igem.org/mediawiki/2016/a/ad/T--Uppsala--design_electroporation_annotated.png"/>
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        <p>
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            The running channels in the electroporation chip are 200 µm across and 100 µm high, while the “channels” where the electrodes are supposed to be placed are 200x200 µm. The inlet channel for the cell suspension is 500 µm wide and 100 µm high. The total distance that the liquid travels inside the resistor is 103.4 mm or about 10 cm. The resistor which we added is theorized to produce longer and larger droplets, since these scale with the length of the outlet channel.(Ref 1.).
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        </p>
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        <h4>References</h4>
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        <p>
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            <small>
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Resistor: http://www.rsc.org/suppdata/lc/b5/b510841a/b510841a.pdf
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Supplemental information to Formation of droplets and bubbles in a microfluidic T-junction—scaling and mechanism of break-up
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Piotr Garstecki, Michael J. Fuerstman, Howard A. Stone and George M. Whitesides
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</small>
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        </p>
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</div>
 
</body>
 
</body>
  

Latest revision as of 12:22, 19 October 2016

Proof of concept

As a proof of concept we did 2 rounds of experiments on our heat shock chip, one where we compared its transformation efficiency to that of regular heat shock transformation done off-chip and one where we compared transformation efficiency on the chip at different temperatures.

Heat Shock Chip

During the summer we made over 30 chips and numerous successful heat shock transformations using only 8.4 ng of DNA and 5.9 μL of competent cells.

Bacterial growth was seen on nearly all of the chip transformed plates. In other words, the transformation worked on our chip and it greatly reduced the amount of reagents needed since colony growth was observed on plates were only 6mL of cell/DNA suspension was heat shocked. The number of colonies for each trial was added by counting all of the five plates for each trial as one replicate. That left three replicates for each chip. Calculations of colony forming units per microliter DNA were made and the mean for each chip is shown in Figure 1. We found that the transformation efficiency was much higher in the conventional heat shock than on our chip.

The plates with bacteria transformed on the chip showed many colonies, considering the small amount of cells and DNA that were plated. A large variation in number of colonies between different trials was observed. That is, two transformations carried out on the chip in the same manner could yield a varying number of colonies.

The cleanliness of our chip was good as little to no colonies grew on the plates with only SOB run through the chip nor on the plates with only cells (negative control).

Figure 1. Mean transformation efficiencies of the different chips (1-3) and conventional transformations (0). Transformation efficiencies calculated by: # colonies on plate/g of DNA plated. Error bars show standard deviation, n=3.



Figure 2. Plates after transformation with chip. Two different trials show the variation in efficiency. Variation could be due to human error.
Raw data

Raw data

Here the raw data of every transformation is presented alongside the comparison transformations we did.

Table 1.
Trial. 34567891011
Chip model123
No. of colonies9641096505911107455857711029
Transformation efficiency (cfu/ng DNA)34.939.618.23338.81.993.0727.937.2


Table 2.
DNA concentration(ng/µl) 4747470.56
No. of Colonies>10001707264
Transformation efficiency (cfu/ng DNA)42.5304129114

According to the results presented in Table 1 and Table 2 the heat shock chip did not reach higher efficiency than conventional methods, at least with the comparison metrics used. There was also very high variation in the transformation efficiency among the experiments done on on the chip(which were done using similar/identical reagents). The efficiency varied from 1,99 in experiment number 8 up to 39,6 in experiment number 4, which is a 20-fold variation in efficiency. On a per plate basis the highest efficiency we had was 123 000 and the lowest was 0 (8 out of 50 plates had 0 colonies), with the average transformation efficiency being 36.

During the experiments we could identify several potential causes which could cause this large variation of results. The setup of the chip involved running heated water from a beaker through the chip and this means that there can be a chance that there is higher or lower thermal leakage than expected, leading to the transformation efficiency being affected. Also we were not sure whether heat shocking at 42 degrees for 35-40 seconds is the optimal way to do it. Since the heat is conducted much better and faster through the chip from the heating channels and because the cells are in a much smaller volume of media the cells are heated more rapidly maybe a lower/higher temperature should be used a shorter/longer time. Furthermore we noticed that there was a very large variance due to the human factor(i.e. who was actually conducting the experiment) and that experience in the procedure played a large role in how successful the transformation was. This was likely due to the primitive and direct way cells were fed into and out of the chip (using a syringe to push in/out cells and eye-measuring to see whether cells have entered the chip makes for inaccurate timing and a lot of back and forth).

Results for the temperature comparisons

For each transformation at a set temperature the amount of formed colonies can be viewed in figure 3. Figure 3 shows the data points for the triplicates at each temperature having a large spread of the amount of formed colonies. Only the transformations at temp. 55 ◦C showed formed colonies for each of the three replicates.

Figure 3. Number of colonies formed after transformation at each temperature. The crosses represent each replicate, the circle is a contamination check, and the rhombs are the average amount of colonies at each temperature.



The highest average value for formed colonies was at 55 ◦C for the heating fluid, giving 30 colonies whilst heat shocking at 65 ◦C and 75 ◦C yielded 21 and 14.7 colonies. The average (avg) number of colonies at each temperature drops slightly when going from 55 ◦C, 65 ◦C to 75 ◦C since the latter two contain replicates with zero number of colonies.

The avg value for the number of colonies at each temperature was used to determine the average transformation efficiency, represented by the cfu/µg value and is presented in Table 3.

Table 3. Average transformation efficiencies at different temperatures.
Temperature (°C)Transformation efficiency (cfu/ng DNA)
554.11
652.88
752.01