Difference between revisions of "Team:Newcastle/Measurement"

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       <p>In order for advances in synthetic biology to be successful it is necessary to develop precise and robust standards for measurements of parts and devices. Biological systems are extremely sensitive to cellular and environmental changes, therefore standardisation of gene expression is essential for the reported measurements to be reliable. The lack of these standards presents a potential limitation to the creation of Sensynova and other genetically engineered devices. To address this issue, we examined variability found between identical genetically engineered devices under different environmental conditions and as a result of using different assembly standards.</p>
 
       <p>In order for advances in synthetic biology to be successful it is necessary to develop precise and robust standards for measurements of parts and devices. Biological systems are extremely sensitive to cellular and environmental changes, therefore standardisation of gene expression is essential for the reported measurements to be reliable. The lack of these standards presents a potential limitation to the creation of Sensynova and other genetically engineered devices. To address this issue, we examined variability found between identical genetically engineered devices under different environmental conditions and as a result of using different assembly standards.</p>
 
        
 
        
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       <h2 class="text-left" style="margin-top: 2%; margin-bottom: 1%; font-family: Rubik">Interlab Devices in Different Contexts</h2>
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       <h2 class="text-left" style="margin-top: 2%; margin-bottom: 1%; font-family: Rubik">Optimum DNA Assembly Standards</h2>
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       <p>We looked at the impact of temperature, pH and media on how high, medium and low expressing devices performed. Tests 2, 5 and 6 were selected from the Interlab Study after initial results were analysed. Single colonies of each transformant were selected and grown in LB+Chl, then washed and diluted to an OD600 of 0.05 in 100 ul media. Transformants were analysed in quadruplicate, with bacteria and media pipetted onto the plate using a pipetting robot for maximum accuracy. Once set up, the plate was incubated at specified temperatures in a plate reader with double orbital shaking, taking readings every 10 min for 24 h. </p>
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       <p>Gibson assembly proved to be the least time-consuming method to manually produce the synthetic GFP reporter gene compared to PhytoBricks and BioBricks. The production of the GFP reporter construct using the Biobricks method was unsuccessful; the multiple steps involved, time consuming methodology and inefficient enzyme activities throughout this process lead to a limited number of parts that were able to be joined. Additionally, it was found that joining the smaller parts; the promoter and RBS was more challenging than joining the larger parts in the manual assembly. The PhytoBricks assembly method successfully produced the GFP reporter construct however it involved more steps and was more time consuming than Gibson assembly. However, a major limitation of this investigation was human error and a difference in user.
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The Gibson assembly construct had a higher GFP expression level after 24 hours, at 7.46x105 AFU/OD600 with the standard error of 6.51x104 AFU/OD600, compared to the PhytoBricks assembly construct which had the final GFP expression level of 2.17x105 AFU/OD600 with the standard error of 6.68x104 AFU/OD600. There was a significant difference in the GFP expression between the PhytoBricks and Gibson Assembly constructs (Mann-Whitney, U=483, n=25,25, p<0.05).</p>
 
        
 
        
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       <h4 class="text-left" style="margin-top: 2%; margin-bottom: 1%; font-family: Rubik">Temperature</h4>
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       <h2 class="text-left" style="margin-top: 2%; margin-bottom: 1%; font-family: Rubik">Optimum Conditions for Gene Expression</h2>
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       <p>Growth in Test 2 was affected differently in LB and SOC (Fig 3). In LB, there appears to be a slight increase in max OD reached as temperature increases, however at an alkaline pH the device appears unable to grow well in higher temperatures. In SOC, there is no distinct pattern in how temperature affects growth, however it is clear that the bacteria grows better as pH is increased, suggesting pH and temperature have a combined effect.</p>
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       <p><b>2.1. Growth Media:</b><br />
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The 5-alpha strain containing the synthetic reporter construct which was grown in LB media, at 37ᵒC and a neutral pH, had a higher final GFP expression level, at 6.18x105 AFU/OD600 with the standard error of 1.69x105 AFU/OD600, compared to when grown in SOC media which was 4.08x104 AFU/OD600 with the standard error of 1.76x104 AFU/OD600 at 24 hours. There was a significant difference in 5-alpha GFP expression between LB media and SOC media over 24 hours (Mann-Whitney, U=854, n=25,25, p<0.001). The GFP expression of the 5-alpha strains grown in both LB and SOC media decreased during the initial 4 hours of incubation, during the lag phase of growth followed by a substantial increase in expression in LB media over the remaining 20 hours and relatively constant expression level for the final 20 hours in SOC media.</p>
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      <img src="https://static.igem.org/mediawiki/2017/a/a9/T--Newcastle--ODtempHLBSOC2.jpeg" class="img-fluid border border-dark rounded mx-auto d-block">
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      <p class="text-center"><b>Figure 3</b> Max OD reached by Test 2 in (A) LB and (B) SOC media over 24 h at 31C, 37C and 43C.</p>     
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      <p style="margin-top: 2%">Fluorescence levels were affected differently at different pH levels as temperature was increased (Figure 4). 37C appears to be optimum temperature for both media, as there are decreases between 37C and 43C at all pH levels apart from pH 7.20 in LB media. However, we can see a dramatic increase in overall max fluorescence levels in both LB and SOC when pH is increased to an alkaline level.</p>
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      <img src="https://static.igem.org/mediawiki/2017/d/da/T--Newcastle--maxFLtemp.jpeg" class="img-fluid border border-dark rounded mx-auto d-block">
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      <p class="text-center"><b>Figure 4</b> Max fluorescence reached by Test 2 in (A) LB and (B) SOC media over 24 h at 31C, 37C and 43C.
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      <p style="margin-top: 2%">For the overall FL:OD, in LB media 37C was the optimum temperature, but in SOC a higher temperature appeared to be more favourable. In both media, the exception was seen at an alkaline pH, where FL:OD increases with temperature in LB, and 37C was optimum in SOC (Figure 5).</p>
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    <p><b>2.2. Temperature:</b><br />
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The GFP expression of 5-alpha grown at 31ᵒC in SOC media at a neutral pH was 1.68x105 AFU/OD600 with the standard error of 6.10x103 AFU/OD600 after 24 hours of incubation. This was higher than the GFP expression in the 5-alpha grown at 37ᵒC and 43ᵒC, which were 4.08x104 AFU/OD600 with the standard error of 7.13x103 AFU/OD600 and 3.94x104 AFU/OD600 with the standard error of 4.38x103 respectively. There was a significant difference in mean GFP expressions of 5-alpha between 31ᵒC, 37ᵒC and 43ᵒC in SOC media over the 24 hours (ANOVA, F2,72=12.10, p<0.001). A post hoc Tukey test indicated that there was no significant difference between the expression at 37ᵒC and 43ᵒC (p>0.05) but the expression and 31ᵒC was significantly different to the other temperatures (p<0.001). The Gibson Assembly construct in 5-alpha also had a higher GFP expression in LB media compared to SOC media during the stationary growth phase at 31ᵒC.</p>
  
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    <img src="https://static.igem.org/mediawiki/2017/0/0f/T--Newcastle--FLODtemp.jpeg" class="img-fluid border border-dark rounded mx-auto d-block">
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      <p><b>2.3. pH:</b><br />
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      <p class="text-center"><b>Figure 5</b> Max FL:OD reached by Test 2 in (A) LB and (B) SOC media over 24 h at 31C, 37C and 43C.
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The 5-alpha E.coli containing the Gibson Assembly construct which was grown in 37ᵒC SOC media at pH 9 had a higher final GFP expression than the 5-alpha grown at pH 7 or pH 5;  final the GFP expression of 5-alpha at pH 9 was 4.12x105 AFU/OD600 with the standard error of, 2.40x104 whereas the GFP expressions for 5-alpha grown at pH 7 and pH 5 were 4.08x104 AFU/OD600 with the standard error of 7.32x103 AFU/OD600 and 2.88x104 AFU/OD600 with the standard error of 4.27x103 AFU/OD600 respectively. There was a significant difference in mean GFP expression levels between 5-alpha grown at pH 5, 7 and 9 over the 24 hours (ANOVA, F2,72=49.88, p<0.001). A post hoc Tukey test indicated that there was no significant difference in the 5-alpha GFP expression between pH 5 and 7 (p>0.05) but 5-alpha at pH 9 had a significantly higher GFP expression than at the lower pHs (p<0.05).<br />
  
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      <h4 class="text-left" style="margin-top: 2%; margin-bottom: 1%; font-family: Rubik">pH</h4>
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Additionally, the 5-alpha E. coli grown in 37ᵒC LB media had a higher GFP expression at pH 9 than the 5-alpha grown in pH 5 and pH 7 LB media; the GFP expression at pH 9 was 9.13x105 AFU/OD600 with the standard error of 3.45x104 AFU/OD600 whereas the GFP expression at pH 5 and 7 in LB media were 6.38x105 AFU/OD600 with the standard error of 2.49x104 AFU/OD600 and 6.18x105 AFU/OD600 with the standard error of 1.68x104 AFU/OD600 respectively.</p>
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      <p>Growth was affected significantly by changes in pH, which can be seen in figure 6. In SOC media, a distinct increase is seen in max OD as pH increases. In LB media the opposite is seen, where as pH increases, max growth decreases.</p>
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      <img src="https://static.igem.org/mediawiki/2017/4/4f/T--Newcastle--maxODpH.jpeg" class="img-fluid border border-dark rounded mx-auto d-block">
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      <p class="text-center"><b>Figure 6</b> Max OD reached by Test 2 over 24 h in (A) LB and (B) SOC media adjusted to varying pH levels.</p>           
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      <p style="margin-top: 2%">Fluorescence was affected by pH particularly in SOC where an increase was seen with an increase in pH; In LB there was less of a pattern seen; increases can be seen at 31C and 37C with increases in pH, however at 43C the max FL is reduced dramatically. The combination of high temperature/pH may have been toxic to the cells. </p>
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      <img src="https://static.igem.org/mediawiki/2017/f/fa/T--Newcastle--maxFLpH.jpeg" class="img-fluid border border-dark rounded mx-auto d-block">
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      <p class="text-center"><b>Figure 7</b> Max FL reached by Test 2 over 24 h in (A) LB and (B) SOC media adjusted to varying pH levels</p>  
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<p style="margin-top: 2%">Despite clear correlations in both growth and fluorescence, there appears to be no distinct pattern in the increase of pH and the overall FL:OD (Figure 8).  </p>
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  <p><b>2.4. Chassis Strain:</b><br />
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The BL21 E. coli strain containing the Gibson Assembly construct had a significantly higher GFP expression level than the 5-alpha and TOP10 strains when incubated in SOC media at 37ᵒC at a neutral pH; the BL21 GFP expression was 1.01x105 AFU/OD600 with the standard error of 3.66x104 AFU/OD600 at 24 hours whereas 5-alpha was 4.08x104 AFU/OD600 with the standard error of 7.32x103 AFU/OD600 and TOP10 was 4.04x104 AFU/OD600 with the standard error of 1.24x104 AFU/OD600 at 24 hours. There was a significant difference in mean GFP expression levels between 5-alpha, BL21 and TOP10 grown at 37ᵒC at a neutral pH in SOC media over the 24 hours (ANOVA, F2,72=15.16, p<0.001). A post hoc Tukey test indicated that there was no significant difference in the GFP expression between 5-alpha and TOP10 (p>0.05) but BL21 had a significantly higher GFP expression than the other strains (p<0.05).
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      <img src="https://static.igem.org/mediawiki/2017/2/2f/T--Newcastle--maxFLODpH.jpeg" class="img-fluid border border-dark rounded mx-auto d-block">
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TOP10 E. coli grown at 43ᵒC, pH 7 in LB had the highest overall level of GFP expression at 1.31x106 AFU/OD600 with the standard error of 8.65x104 AFU/OD600 which was during the stationary phase of growth at 20 hours. The second highest overall level of GFP was expressed by BL21 at pH 9 in 37ᵒC SOC media at 1.27x106 AFU/OD600 with the standard error of 7.24x104 AFU/OD600.
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      <p class="text-center"><b>Figure 8</b> Max FL:OD reached by Test 2 over 24 h in (A) LB and (B) SOC media adjusted to varying pH levels</p>    
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</p>
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      <p style="margin-top: 2%">Overall, it is clear pH and temperature have an impact on the growth and fluorescence of the devices, and this raises the importance of maintaining conditions specified in InterLab instructions. It is difficult to draw definite conclusions without more thorough experimentation. It should also be noted that whilst each replicate of isolates was added to the plate using a robot for maximum accuracy, there was still a degree of variability. These variances will have increased over time, and the InterLab method of taking samples at specific time points may be more laborious, but will yield a more accurate result of overall culture behaviour.</p>
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      <h2 class="text-left" style="margin-top: 2%; margin-bottom: 1%; font-family: Rubik">Standard Assembly Methods</h2>
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      <p>Gibson assembly proved to be the least time-consuming method to manually produce the synthetic GFP reporter gene compared to PhytoBricks and BioBricks. The production of the GFP reporter construct using the Biobricks method was unsuccessful; the multiple steps involved, time consuming methodology and inefficient enzyme activities throughout this process lead to a limited number of parts that were able to be joined. Additionally, it was found that joining the smaller parts; the promoter and RBS was more challenging than joining the larger parts in the manual assembly. The PhytoBricks assembly method successfully produced the GFP reporter construct however it involved more steps and was more time consuming than Gibson assembly. However, a major limitation of this investigation was human error and a difference in user.
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The Gibson assembly construct had a higher GFP expression level after 24 hours, at 7.46x105 AFU/OD600 with the standard error of 6.51x104 AFU/OD600, compared to the PhytoBricks assembly construct which had the final GFP expression level of 2.17x105 AFU/OD600 with the standard error of 6.68x104 AFU/OD600. There was a significant difference in the GFP expression between the PhytoBricks and Gibson Assembly constructs (Mann-Whitney, U=483, n=25,25, p<0.05).</p>
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      <h2 class="text-left" style="margin-top: 2%; margin-bottom: 1%; font-family: Rubik">Standard Measurement Methods</h2>
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      <p>Text goes here.</p>
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       <h2 class="text-left" style="margin-top: 2%; margin-bottom: 1%; font-family: Rubik">Internal Controls</h2>
 
       <h2 class="text-left" style="margin-top: 2%; margin-bottom: 1%; font-family: Rubik">Internal Controls</h2>

Revision as of 16:02, 31 October 2017

spacefill

Measurement Award

BioBricks used: BBa_J364001, BBa_J364004, BBa_J364005

Rationale and Aim

Text goes here.

Background Information

In order for advances in synthetic biology to be successful it is necessary to develop precise and robust standards for measurements of parts and devices. Biological systems are extremely sensitive to cellular and environmental changes, therefore standardisation of gene expression is essential for the reported measurements to be reliable. The lack of these standards presents a potential limitation to the creation of Sensynova and other genetically engineered devices. To address this issue, we examined variability found between identical genetically engineered devices under different environmental conditions and as a result of using different assembly standards.

Optimum DNA Assembly Standards

Gibson assembly proved to be the least time-consuming method to manually produce the synthetic GFP reporter gene compared to PhytoBricks and BioBricks. The production of the GFP reporter construct using the Biobricks method was unsuccessful; the multiple steps involved, time consuming methodology and inefficient enzyme activities throughout this process lead to a limited number of parts that were able to be joined. Additionally, it was found that joining the smaller parts; the promoter and RBS was more challenging than joining the larger parts in the manual assembly. The PhytoBricks assembly method successfully produced the GFP reporter construct however it involved more steps and was more time consuming than Gibson assembly. However, a major limitation of this investigation was human error and a difference in user. The Gibson assembly construct had a higher GFP expression level after 24 hours, at 7.46x105 AFU/OD600 with the standard error of 6.51x104 AFU/OD600, compared to the PhytoBricks assembly construct which had the final GFP expression level of 2.17x105 AFU/OD600 with the standard error of 6.68x104 AFU/OD600. There was a significant difference in the GFP expression between the PhytoBricks and Gibson Assembly constructs (Mann-Whitney, U=483, n=25,25, p<0.05).

Optimum Conditions for Gene Expression

2.1. Growth Media:
The 5-alpha strain containing the synthetic reporter construct which was grown in LB media, at 37ᵒC and a neutral pH, had a higher final GFP expression level, at 6.18x105 AFU/OD600 with the standard error of 1.69x105 AFU/OD600, compared to when grown in SOC media which was 4.08x104 AFU/OD600 with the standard error of 1.76x104 AFU/OD600 at 24 hours. There was a significant difference in 5-alpha GFP expression between LB media and SOC media over 24 hours (Mann-Whitney, U=854, n=25,25, p<0.001). The GFP expression of the 5-alpha strains grown in both LB and SOC media decreased during the initial 4 hours of incubation, during the lag phase of growth followed by a substantial increase in expression in LB media over the remaining 20 hours and relatively constant expression level for the final 20 hours in SOC media.

2.2. Temperature:
The GFP expression of 5-alpha grown at 31ᵒC in SOC media at a neutral pH was 1.68x105 AFU/OD600 with the standard error of 6.10x103 AFU/OD600 after 24 hours of incubation. This was higher than the GFP expression in the 5-alpha grown at 37ᵒC and 43ᵒC, which were 4.08x104 AFU/OD600 with the standard error of 7.13x103 AFU/OD600 and 3.94x104 AFU/OD600 with the standard error of 4.38x103 respectively. There was a significant difference in mean GFP expressions of 5-alpha between 31ᵒC, 37ᵒC and 43ᵒC in SOC media over the 24 hours (ANOVA, F2,72=12.10, p<0.001). A post hoc Tukey test indicated that there was no significant difference between the expression at 37ᵒC and 43ᵒC (p>0.05) but the expression and 31ᵒC was significantly different to the other temperatures (p<0.001). The Gibson Assembly construct in 5-alpha also had a higher GFP expression in LB media compared to SOC media during the stationary growth phase at 31ᵒC.

2.3. pH:
The 5-alpha E.coli containing the Gibson Assembly construct which was grown in 37ᵒC SOC media at pH 9 had a higher final GFP expression than the 5-alpha grown at pH 7 or pH 5; final the GFP expression of 5-alpha at pH 9 was 4.12x105 AFU/OD600 with the standard error of, 2.40x104 whereas the GFP expressions for 5-alpha grown at pH 7 and pH 5 were 4.08x104 AFU/OD600 with the standard error of 7.32x103 AFU/OD600 and 2.88x104 AFU/OD600 with the standard error of 4.27x103 AFU/OD600 respectively. There was a significant difference in mean GFP expression levels between 5-alpha grown at pH 5, 7 and 9 over the 24 hours (ANOVA, F2,72=49.88, p<0.001). A post hoc Tukey test indicated that there was no significant difference in the 5-alpha GFP expression between pH 5 and 7 (p>0.05) but 5-alpha at pH 9 had a significantly higher GFP expression than at the lower pHs (p<0.05).
Additionally, the 5-alpha E. coli grown in 37ᵒC LB media had a higher GFP expression at pH 9 than the 5-alpha grown in pH 5 and pH 7 LB media; the GFP expression at pH 9 was 9.13x105 AFU/OD600 with the standard error of 3.45x104 AFU/OD600 whereas the GFP expression at pH 5 and 7 in LB media were 6.38x105 AFU/OD600 with the standard error of 2.49x104 AFU/OD600 and 6.18x105 AFU/OD600 with the standard error of 1.68x104 AFU/OD600 respectively.

2.4. Chassis Strain:
The BL21 E. coli strain containing the Gibson Assembly construct had a significantly higher GFP expression level than the 5-alpha and TOP10 strains when incubated in SOC media at 37ᵒC at a neutral pH; the BL21 GFP expression was 1.01x105 AFU/OD600 with the standard error of 3.66x104 AFU/OD600 at 24 hours whereas 5-alpha was 4.08x104 AFU/OD600 with the standard error of 7.32x103 AFU/OD600 and TOP10 was 4.04x104 AFU/OD600 with the standard error of 1.24x104 AFU/OD600 at 24 hours. There was a significant difference in mean GFP expression levels between 5-alpha, BL21 and TOP10 grown at 37ᵒC at a neutral pH in SOC media over the 24 hours (ANOVA, F2,72=15.16, p<0.001). A post hoc Tukey test indicated that there was no significant difference in the GFP expression between 5-alpha and TOP10 (p>0.05) but BL21 had a significantly higher GFP expression than the other strains (p<0.05). TOP10 E. coli grown at 43ᵒC, pH 7 in LB had the highest overall level of GFP expression at 1.31x106 AFU/OD600 with the standard error of 8.65x104 AFU/OD600 which was during the stationary phase of growth at 20 hours. The second highest overall level of GFP was expressed by BL21 at pH 9 in 37ᵒC SOC media at 1.27x106 AFU/OD600 with the standard error of 7.24x104 AFU/OD600.

Internal Controls

Text goes here.

Robust Promoter Characterisation

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Conclusions and Future Work

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References

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