Difference between revisions of "Team:Munich/Testing/celllysis"

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<h2> Cell Lysis </h2> </br></br>
 
<h2> Cell Lysis </h2> </br></br>
 
<img src="https://static.igem.org/mediawiki/2017/thumb/4/48/T--Munich--pic--lysis_alkaline_degradation.png/541px-T--Munich--pic--lysis_alkaline_degradation.png">
 
<img src="https://static.igem.org/mediawiki/2017/thumb/4/48/T--Munich--pic--lysis_alkaline_degradation.png/541px-T--Munich--pic--lysis_alkaline_degradation.png">
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From our Urea page gel, we conclude that alkaline lysis, as frequently used for plasmid extraction, is a very fast way of extracting RNA. It is commonly known that RNA (unlike DNA) swiftly self-degredates at high pH, and this also showed in our experiments. Comparison between different incubation times shows clearly the continuing degredation over time. Within only a few seconds of inverting tubes, RNA is already cut and thus shortened, as the two "NaOH, 0 min" bars show. However, the target sequence of Cas13a is very short (~ 20 base pairs) and we deem it unlikely that a significant percentage of target-sequences is destroyed by a very fast lysis. Most of the cut strands are still well above 200 bp for a "seconds-long" lysis, with the peak of brightness between the 500 bp and 1000 bp mark, as the gradient of band intensity shows. With higher incubation times, the peak of brightness goes steadily lower, and even under 200 bp for a incubation time of 10 minutes. It is thus of utmost importance to keep the lysis time to a few seconds, which makes a proper and fast mixing of the sample with alkalic lysis buffer and seconds later with acidic equilibration buffer necessary. Since microfluidic mixing of liquids is a rather complex process, we chose a tecnically simpler approach to extract pathogen RNA in our first prototype: Isothermal PCR.
 
From our Urea page gel, we conclude that alkaline lysis, as frequently used for plasmid extraction, is a very fast way of extracting RNA. It is commonly known that RNA (unlike DNA) swiftly self-degredates at high pH, and this also showed in our experiments. Comparison between different incubation times shows clearly the continuing degredation over time. Within only a few seconds of inverting tubes, RNA is already cut and thus shortened, as the two "NaOH, 0 min" bars show. However, the target sequence of Cas13a is very short (~ 20 base pairs) and we deem it unlikely that a significant percentage of target-sequences is destroyed by a very fast lysis. Most of the cut strands are still well above 200 bp for a "seconds-long" lysis, with the peak of brightness between the 500 bp and 1000 bp mark, as the gradient of band intensity shows. With higher incubation times, the peak of brightness goes steadily lower, and even under 200 bp for a incubation time of 10 minutes. It is thus of utmost importance to keep the lysis time to a few seconds, which makes a proper and fast mixing of the sample with alkalic lysis buffer and seconds later with acidic equilibration buffer necessary. Since microfluidic mixing of liquids is a rather complex process, we chose a tecnically simpler approach to extract pathogen RNA in our first prototype: Isothermal PCR.
 
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<img src="https://static.igem.org/mediawiki/2017/thumb/6/6e/T--Munich--pic--lysis_pcr_deltatemp.png/800px-T--Munich--pic--lysis_pcr_deltatemp.png"> </br>
 
<img src="https://static.igem.org/mediawiki/2017/thumb/6/6e/T--Munich--pic--lysis_pcr_deltatemp.png/800px-T--Munich--pic--lysis_pcr_deltatemp.png"> </br>
 
[1] 65 °C, [2] log2 DNA ladder, [3] 70 °C, [4] 75 °C, [5] 80 °C, [6] 85 °C, [7] 90 °C, [8] empty °C, [9] positive control (DNA sequence diluted), [10] log2 DNA ladder </br>
 
[1] 65 °C, [2] log2 DNA ladder, [3] 70 °C, [4] 75 °C, [5] 80 °C, [6] 85 °C, [7] 90 °C, [8] empty °C, [9] positive control (DNA sequence diluted), [10] log2 DNA ladder </br>
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To increase the detection threshold of our system with PCR, it is however still important to optimize the heat lysis parameters, most prominently the temperature at which to incubate. It has been shown, at least for protein extraction <verweis literatur todo>, that higher temperature not necessarily leads to better lysis efficiency. Thats why we tested isothermal PCR (RPA) on samples lysed with different temperatures (65 °C, 70 °C, 75 °C, 80 °C, 85 °C, 90 °C) and concluded from our RPA gel that any temperature above 75 °C degree works at least as good as 90 °C, if not better. This is consistent to mentioned literature and allows us to use lower temperatures in our device, which reduces issues with evaporation and build-up pressure. Still, a higher temperature of  over 90 °C might be advantageous to reduce RNAse activity.
 
To increase the detection threshold of our system with PCR, it is however still important to optimize the heat lysis parameters, most prominently the temperature at which to incubate. It has been shown, at least for protein extraction <verweis literatur todo>, that higher temperature not necessarily leads to better lysis efficiency. Thats why we tested isothermal PCR (RPA) on samples lysed with different temperatures (65 °C, 70 °C, 75 °C, 80 °C, 85 °C, 90 °C) and concluded from our RPA gel that any temperature above 75 °C degree works at least as good as 90 °C, if not better. This is consistent to mentioned literature and allows us to use lower temperatures in our device, which reduces issues with evaporation and build-up pressure. Still, a higher temperature of  over 90 °C might be advantageous to reduce RNAse activity.
 
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<img src="https://static.igem.org/mediawiki/2017/thumb/e/e0/T--Munich--pic--lysis_pcr_deltacells.png/800px-T--Munich--pic--lysis_pcr_deltacells.png"> </br>
 
<img src="https://static.igem.org/mediawiki/2017/thumb/e/e0/T--Munich--pic--lysis_pcr_deltacells.png/800px-T--Munich--pic--lysis_pcr_deltacells.png"> </br>
 
[1] log2 DNA ladder, [2] 10^6 cells/ml, [3] 10^5 cells/ml, [4] 5*10^4 cells/ml, [5] 10^4 cells/ml,[6] 5*10^3 cells/ml, [7] 10^3 cells/ml, [8] 10^2 cells/ml,[9] 10^6 cells/ml (no heat-step. only PCR at 37 °C)
 
[1] log2 DNA ladder, [2] 10^6 cells/ml, [3] 10^5 cells/ml, [4] 5*10^4 cells/ml, [5] 10^4 cells/ml,[6] 5*10^3 cells/ml, [7] 10^3 cells/ml, [8] 10^2 cells/ml,[9] 10^6 cells/ml (no heat-step. only PCR at 37 °C)

Revision as of 11:26, 28 October 2017

Cell Lysis



From our Urea page gel, we conclude that alkaline lysis, as frequently used for plasmid extraction, is a very fast way of extracting RNA. It is commonly known that RNA (unlike DNA) swiftly self-degredates at high pH, and this also showed in our experiments. Comparison between different incubation times shows clearly the continuing degredation over time. Within only a few seconds of inverting tubes, RNA is already cut and thus shortened, as the two "NaOH, 0 min" bars show. However, the target sequence of Cas13a is very short (~ 20 base pairs) and we deem it unlikely that a significant percentage of target-sequences is destroyed by a very fast lysis. Most of the cut strands are still well above 200 bp for a "seconds-long" lysis, with the peak of brightness between the 500 bp and 1000 bp mark, as the gradient of band intensity shows. With higher incubation times, the peak of brightness goes steadily lower, and even under 200 bp for a incubation time of 10 minutes. It is thus of utmost importance to keep the lysis time to a few seconds, which makes a proper and fast mixing of the sample with alkalic lysis buffer and seconds later with acidic equilibration buffer necessary. Since microfluidic mixing of liquids is a rather complex process, we chose a tecnically simpler approach to extract pathogen RNA in our first prototype: Isothermal PCR.


[1] 65 °C, [2] log2 DNA ladder, [3] 70 °C, [4] 75 °C, [5] 80 °C, [6] 85 °C, [7] 90 °C, [8] empty °C, [9] positive control (DNA sequence diluted), [10] log2 DNA ladder
2 uL of lysed E. Coli suspension with density of 10^6 cells/ml used for PCR-Mix.

PCR is a common method in analytics to achieve huge amplification of genetic material. It allows us to use a relatively inefficient lysis method, lysis by heat only, and increases the resulting low concentration of target sequence to above a detectable threshold. With Reverse Transcription before, it is also possible to amplify an RNA sequence as DNA.
To increase the detection threshold of our system with PCR, it is however still important to optimize the heat lysis parameters, most prominently the temperature at which to incubate. It has been shown, at least for protein extraction , that higher temperature not necessarily leads to better lysis efficiency. Thats why we tested isothermal PCR (RPA) on samples lysed with different temperatures (65 °C, 70 °C, 75 °C, 80 °C, 85 °C, 90 °C) and concluded from our RPA gel that any temperature above 75 °C degree works at least as good as 90 °C, if not better. This is consistent to mentioned literature and allows us to use lower temperatures in our device, which reduces issues with evaporation and build-up pressure. Still, a higher temperature of over 90 °C might be advantageous to reduce RNAse activity.


[1] log2 DNA ladder, [2] 10^6 cells/ml, [3] 10^5 cells/ml, [4] 5*10^4 cells/ml, [5] 10^4 cells/ml,[6] 5*10^3 cells/ml, [7] 10^3 cells/ml, [8] 10^2 cells/ml,[9] 10^6 cells/ml (no heat-step. only PCR at 37 °C)

To assess the theoretical sensibility of our PCR-enhanced lysis, we did tests on a dilution series of a E. coli with TEV-plasmid (psB1C3-His-008-TEV) culture with a cell density range from 10^6 to 10^2 cells/ml. After lysis at 80 °C, the isothermal PCR was performed on 2 uL of lysed sample. The gel shows a clearly visible band of PCR product down to a cell density of 5*10^4 cells/ml. Image processing even reveals low PCR-product concentration at 10^4 cell/ml, which corresponds to on average 20 cells in the PCR reaction volume. This fits nicely with our findings that heat-only lysis is less efficient by a factor of 5 to 10 when compared to lysis with detergents and heat (see labbook).