Difference between revisions of "Part:BBa K914003:Experience"

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how you used this part and how it worked out.
 
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===Applications of BBa_K914003===
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===Applications of BBa_M50055===
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This part is intended to be an atrazine biosensor that uses GFP expression to reflect atrazine levels in the surrounding environment. However, we found that it is also sensitive to simazine, which is chemically similar to atrazine, indicating that this plasmid could potentially be used as a broad-application biosensor for triazine class pesticides.
  
 
===User Reviews===
 
===User Reviews===
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<partinfo>BBa_K914003 AddReview number</partinfo>
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<I>Wageningen UR 2014</I>
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We used this part in our project and characetrized it using gfp.  
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Enter the review inofrmation here.
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https://static.igem.org/mediawiki/2014/f/f9/Wageningen_UR_killswitch_rhamnose.png <br>
 
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''Figure 1. Average GFP fluorescence of E. coli DH5α carrying a pSB3K3 plasmid containing the pRha gfp BioBrick. At t=0 the cells were induced with different concentrations of rhamnose in triplo. Fluorescence is measured in a plate reader in time (A) and at time point 8.25 (B) normalized for OD600.''
 
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The measurements in Figure 1 indicate a tuneable activation of pRha by rhamnose. The RFU values of 0% and 0.001% rhamnose are not significant taking into account the high standard deviation for these measurements as can be seen in Figure 1B. From a rhamnose concentration of 0.01% to 0.2% a significant increase in fluorescence is measured. Fluorescence from Figure 2 can be used to predict the concentration of repressor protein produced. Data points for time 8.25 h were chosen for the graph in Figure 2B due to the peak at time for 0.2% rhamnose, which is visible in Figure 1. This peak can be explained by the rhamnose depletion, as it is consumed by E. coli, causing the stop of promoter induction. We conclude that the L-rhamnose inducible promoter part BBa_K914003 is a well functional part. 
 
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We ordered this part from DNA 2.0 with the addition of an IPTG-inducible promoter. The plasmid was then transformed into E. coli and cultured in LB + kanamycin.
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We first conducted an assay to see the effects of IPTG, atrazine, and simazine on GFP expression. 0.5 mL of E.coli cells containing the BBa_M50055 plasmid at an OD600 of 0.01 was incubated with the following concentrations of IPTG: 0mM, 0.1 mM, 0.2 mM, 0.5 mM, and 1 mM, and atrazine: 0 μM, 10 μM, 20 μM, 50 μM, and 100 μM, for a total of 25 different IPTG-atrazine concentration combinations. An equivalent assay was conducted with simazine in place of atrazine to assess the aptamer’s selectivity.
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Fold expression was calculated by comparing the ratio of lowest GFP fluorescence, which we hypothesized would occur at lower combinations of lower concentrations of atrazine (0 μM) and IPTG (0 mM), to that of different IPTG and atrazine concentrations. Our results demonstrate that different concentration combinations of IPTG and atrazine produce unique GFP expression levels that are dose- dependent with both IPTG and atrazine. GFP expression begins to become pronounced abruptly at higher IPTG (0.5 mM) and lower atrazine (10 μM) concentrations, and quickly demonstrates saturation at higher atrazine concentrations, implying a low limit of detection. Interestingly, the bacterial biosensors also reacted with simazine, although the GFP expression levels were notably lower than those observed for atrazine. These results indicate a slight cross-reactivity to simazine, most likely due to the chemical similarity of simazine and atrazine. In light of this data, the developed plasmid could serve as a broad- application biosensor for triazine class pesticides.
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The figures below show the expression trajectories of our riboswitch based on atrazine (A) and the negative control simazine (B). Optical density (600 nm) and GFP expression (excitation filter: 485 + 20 nm; emission filter 535 + 25 nm at a gain of 75) were measured with a plate reader after 24 hours of growth. Output data was produced by normalizing GFP expression with the OD600. All expression assays were composed of three samples for each concentration combination for a total of 75 samples per assay and each type of assay was replicated two additional times to enable statistical analysis.
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[[File:atrazine heatmap.jpg]]
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[[File:simazine heatmap.jpg]]
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The average GFP/OD600 value for the blank media negative control was 210 with a standard deviation of 54. The average GFP/OD600 value for E.coli transformed with pHeLa was 64219 with a standard deviation of 801.
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We ran a second assay to determine the sensitivity of our biosensor. We grew experimental cultures in LG +kanamycin as described above with 1 mM of IPTG, to fully transcribe the developed riboswitch, along with serial dilutions of atrazine (0, 0.01, 0.02, 0.05, 0.1, 0.2, 0.5, 1, 2, 5, 10, 20, 50, 100 μM). We then employed the GFP assay from above, with identical controls, and each measurement was performed in triplicate and two replicates were conducted to enable statistical analysis. We found the biosensor elicited a strong dose-dependent response to atrazine in the dynamic range of 0.05-2 μM. Linear least- squares of the logarithmically adjusted data in the dynamic range gave a coefficient of determination (R2) of 0.9767. However, for greater concentrations of atrazine, the response deviated from the linear regression, eventually plateauing at approximately 20 μM, which is indicative of the saturation phenomena when most of the transcribed riboswitches were bound to the target analyte. The concentration that we observed saturation was lower than expected, which we hypothesize was due to the inhibition of translation as a result of the bound structure of the riboswitch still impeding binding at the RBS. The biosensor demonstrated an excellent limit of detection of 39 nM for atrazine, which we calculated using the formula LOD = 3.3SD/S, where SD is the average standard deviation and S is the slope of the linear regression in the dynamic range. Although this LOD is slightly higher than the EPA’s maximum contaminant level of 12 nM, the aptamer design could be further developed to improve the sensitivity of the biosensor. Unfortunately, due to time constraints, we could not assess simazine and this will be left for future work.
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Revision as of 04:11, 12 June 2018


This experience page is provided so that any user may enter their experience using this part.
Please enter how you used this part and how it worked out.

Applications of BBa_M50055

This part is intended to be an atrazine biosensor that uses GFP expression to reflect atrazine levels in the surrounding environment. However, we found that it is also sensitive to simazine, which is chemically similar to atrazine, indicating that this plasmid could potentially be used as a broad-application biosensor for triazine class pesticides.

User Reviews

UNIQdb4555f44fafba9c-partinfo-00000000-QINU UNIQdb4555f44fafba9c-partinfo-00000001-QINU


We ordered this part from DNA 2.0 with the addition of an IPTG-inducible promoter. The plasmid was then transformed into E. coli and cultured in LB + kanamycin.

We first conducted an assay to see the effects of IPTG, atrazine, and simazine on GFP expression. 0.5 mL of E.coli cells containing the BBa_M50055 plasmid at an OD600 of 0.01 was incubated with the following concentrations of IPTG: 0mM, 0.1 mM, 0.2 mM, 0.5 mM, and 1 mM, and atrazine: 0 μM, 10 μM, 20 μM, 50 μM, and 100 μM, for a total of 25 different IPTG-atrazine concentration combinations. An equivalent assay was conducted with simazine in place of atrazine to assess the aptamer’s selectivity.

Fold expression was calculated by comparing the ratio of lowest GFP fluorescence, which we hypothesized would occur at lower combinations of lower concentrations of atrazine (0 μM) and IPTG (0 mM), to that of different IPTG and atrazine concentrations. Our results demonstrate that different concentration combinations of IPTG and atrazine produce unique GFP expression levels that are dose- dependent with both IPTG and atrazine. GFP expression begins to become pronounced abruptly at higher IPTG (0.5 mM) and lower atrazine (10 μM) concentrations, and quickly demonstrates saturation at higher atrazine concentrations, implying a low limit of detection. Interestingly, the bacterial biosensors also reacted with simazine, although the GFP expression levels were notably lower than those observed for atrazine. These results indicate a slight cross-reactivity to simazine, most likely due to the chemical similarity of simazine and atrazine. In light of this data, the developed plasmid could serve as a broad- application biosensor for triazine class pesticides.

The figures below show the expression trajectories of our riboswitch based on atrazine (A) and the negative control simazine (B). Optical density (600 nm) and GFP expression (excitation filter: 485 + 20 nm; emission filter 535 + 25 nm at a gain of 75) were measured with a plate reader after 24 hours of growth. Output data was produced by normalizing GFP expression with the OD600. All expression assays were composed of three samples for each concentration combination for a total of 75 samples per assay and each type of assay was replicated two additional times to enable statistical analysis.

Atrazine heatmap.jpg
Simazine heatmap.jpg
The average GFP/OD600 value for the blank media negative control was 210 with a standard deviation of 54. The average GFP/OD600 value for E.coli transformed with pHeLa was 64219 with a standard deviation of 801.


We ran a second assay to determine the sensitivity of our biosensor. We grew experimental cultures in LG +kanamycin as described above with 1 mM of IPTG, to fully transcribe the developed riboswitch, along with serial dilutions of atrazine (0, 0.01, 0.02, 0.05, 0.1, 0.2, 0.5, 1, 2, 5, 10, 20, 50, 100 μM). We then employed the GFP assay from above, with identical controls, and each measurement was performed in triplicate and two replicates were conducted to enable statistical analysis. We found the biosensor elicited a strong dose-dependent response to atrazine in the dynamic range of 0.05-2 μM. Linear least- squares of the logarithmically adjusted data in the dynamic range gave a coefficient of determination (R2) of 0.9767. However, for greater concentrations of atrazine, the response deviated from the linear regression, eventually plateauing at approximately 20 μM, which is indicative of the saturation phenomena when most of the transcribed riboswitches were bound to the target analyte. The concentration that we observed saturation was lower than expected, which we hypothesize was due to the inhibition of translation as a result of the bound structure of the riboswitch still impeding binding at the RBS. The biosensor demonstrated an excellent limit of detection of 39 nM for atrazine, which we calculated using the formula LOD = 3.3SD/S, where SD is the average standard deviation and S is the slope of the linear regression in the dynamic range. Although this LOD is slightly higher than the EPA’s maximum contaminant level of 12 nM, the aptamer design could be further developed to improve the sensitivity of the biosensor. Unfortunately, due to time constraints, we could not assess simazine and this will be left for future work.