Team:KULeuven/Results

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KULeuven iGEM 2011

Results

1. Promotor testing of inducible promotors

pBAD + GFP generator (Part:BBa_K584000)

To test the usefulness of the arabinose-inducible promoter I13453 in our 2011 iGEM project, we fused the promoter to a GFP reporter, and assayed the promoter’s activity after addition of different amounts of arabinose. We tested this activity both in a TOP10F’ (figure 1) as well as a MG1655 (figure 2) ''E.coli'' strain background. For more information on ''E.coli'' strain descriptions, we recommend the following web site: http://openwetware.org/wiki/E._coli_genotypes. As can be seen in figure 1, addition of arabinose had only minor, if any, effect on the growth of TOP10F’ cells, most likely because these cells cannot metabolize the sugar. However, to our surprise, we did not observe an arabinose-induced increase of fluorescence compared to the control without arabinose for the first 6 hours. The observed increase in fluorescence after arabinose addition at the 8 hour time point could not be confirmed when the experiment was repeated. Figure 2 demonstrates that in MG1655 cells, the addition of 0.2% and 2% arabinose results in a growth defect after about 4 hours of growth, which may relate to the metabolism of the sugar in this strain. In contrast to the TOP10F’ cells, we see a clear induction of promoter activity after addition of arabinose. The addition of only 0.02% resulted in the greatest induction. Increasing arabinose concentration did not increase fluorescence, probably due to the observed growth defect. As an additional control, we checked the activity of a constitutive promoter under the same conditions as described here. For results on these experiments, check out our BBa_K584001 page.

Constitutive promotor + GFP generator (Part:BBa_K584001)

We fused the constitutive promoter to a GFP reporter, and assayed the promoter’s activity after addition of different amounts of arabinose and IPTG. As such these results can serve as a control for the results obtained for the arabinose-inducible BBa_K584000 and IPTG-inducible BBa_K584002 bricks. We tested the activity both in a TOP10F’ (figure 1 & 3) as well as a MG1655 (figure 2 & 4) ''E.coli'' strain background. For more information on ''E.coli'' strain descriptions, we recommend the following web site: http://openwetware.org/wiki/E._coli_genotypes. Apart from a small growth defect and somewhat lower fluorescence at the 8 hour time point after the addition of 2% arabinose, the activity of the constitutive promoter in TOP10F’ cells is unaffected by arabinose addition, as seen in Figure1. For the MG1655 cells (Figure 2), however, we see clear effects of adding arabinose at concentrations of 0.2% or 2%, with promoter activities significantly lower than the control condition. This points to interference of high inducer levels with the activity of the promoter and the importance of metabolism of the inducer, as arabinose can be metabolized by MG1655 cells, but not by TOP10F’ cells.

pLac-Lux hybrid + GFP generator (Part:BBa_K584002)

To gain insights into the usefulness of the lactose-inducible promoter I13453 in our 2011 iGEM project, we fused the promoter to a GFP reporter, and assayed the promoter’s activity after addition of different amounts of IPTG. IPTG is a molecular mimic of allolactose, a lactose metabolite that triggers transcription of the lac operon. Unlike lactose, IPTG cannot be metabolized by wild-type ''E.coli'' cells, leading to a constitutive high presence of inductor. For this reason, IPTG is often used instead of lactose to induce the lac operon. We tested the activity both in a TOP10F’ (figure 1) as well as a MG1655 (figure 2) ''E.coli'' strain background. For more information on ''E.coli'' strain descriptions, we recommend the following web site: http://openwetware.org/wiki/E._coli_genotypes. Addition of IPTG to TOP10F’ cells results in a minor growth defect, as can be seen in Figure 1A. However, this does not seem to inhibit the induction of the promoter, as Figure 1B clearly demonstrates that IPTG results in a clear induction of fluorescence, while without IPTG no such induction is seen. In MG1655 ''E.coli'' cells, IPTG at a concentration of 1.5mM leads to a minor growth defect (Figure 2A). Looking at the fluorescence measurements (Figure 2B), it is clear that the promoter already displays high activity in the absence of inducer, and that this activity is not increased by adding IPTG. More still, adding IPTG at concentrations of 1mM or 1.5mM even results in a lower fluorescence, and hence promoter activity, than the situation without IPTG addition. We think that this is due to the fact that we express our construct on a multicopy vector, which may outcompete the inhibitory activity of the LacI repressor under these conditions. In contrast to the MG1655 cells, the TOP10F’ strain contains a high copy LacI repressor, making the investigated promoter activity not leaky. These experiments highlight the importance of checking the strain background for compatibility with a desired system. '' The investigated promoter, pLac-Lux (I13453), should also be repressed by the luxR repressor bound to the corepressor CO6HSL. Due to time limitations, we could not investigate this aspect of the promoter. As an additional control, we checked the activity of a constitutive promoter under the same conditions as described here. For results on these experiments, check out our BBa_K584001 page.

HybB promotor + GFP generator (Part:BBa_K584004 )

To test the usefulness of the cold shock-inducible promoter J45503 in our 2011 iGEM project, we fused the promoter to a GFP reporter, and assayed the promoter’s activity after a temperature shift from 37°C to 25°C or 4°C. We tested this activity both in a TOP10F’ (figure 1) as well as a MG1655 (figure 2) ''E.coli'' strain background. For more information on ''E.coli'' strain descriptions, we recommend the following web site: http://openwetware.org/wiki/E._coli_genotypes. We can see clearly that transferring cells (both TOP10F’ and MG1655) to lower temperatures (4°C and even 25°C) results in a growth arrest between the 1 and 4 hour time points of our experiment (Figures 1A and 2A). We see that promoter activity is induced when cells are transferred to 25°C and even when they are put in and ice bath (4°C) (Figures 1B and 2B). Unfortunately, however, cells that are kept at 37°C also display an increase in promoter activity, indicating leakiness in the system.

2. Qualitative measurements of INP and AFP activity

First of all, four different types of water were tested to see which one showed the least fluctuations in ice nucleation temperature and thus could be used for an optimal initial characterisation of INP and AFP. To do this, we measured the temperature at which ice nucleation in 25mL of tap water, Milli-Q, deionized water and filtered Milli-Q was induced. An ethanol bath was set at 0°C and falcons containing 25ml of the different types of water were added. The temperature was then gradually cooled to -30°C. This experiment was performed in triple and results showed that deionized water had the smallest fluctuations in induction of ice nucleation (Figure 1). Deionized water will thus be used for all further experiments. It should be noted that this is no quantitative approach as the temperature in the falcons was not monitored, but only the temperature of the ethanol bath. To see whether the time necessary for the induction of ice nucleation was influenced by AFP and INP, an ethanol bath was set at - 30°C and falcons containing 25mL of deionized water with no cells, cells expressing a GFP, the INP or the AFP construct were added. The time necessary for ice nucleation was measured and the experiment was done in triple and repeated two times. Figure 2 shows that the time necessary to induce ice nucleation is vastly reduced when cells expressing INP are added, whereas the time increased when cells expressing AFP were added. The speed of ice nucleation of deionized water without cells or with cells expressing a GFP construct was only marginally different. Another experiment was carried out to learn if the cells expressing AFP or INP could be used multiple times. Ice nucleation was induced in 25mL of deionized water containing cells expressing either AFP or INP, the formed ice was then thawed and the same water was used to re-induce ice nucleation. The time necessary to re-induce ice nucleation was not strikingly different from the first ice nucleation induction. The experiment was performed in triple and the data are shown in figure 2. In conclusion, both AFP and INP can be re-used without loss of activity. Finally, we checked if INP or AFP expression influences the speed of ice thawing. This was performed by freezing 25mL of deionized water at -30°C containing no cells, cells expressing a GFP, the INP or AFP construct. Subsequently the falcons were put in a water bath at 21°C and the time necessary for thawing was measured. This experiment was done in triple and, as can be seen in figure 3, all the different solutions thawed at a similar speed. Hence, we conclude that neither AFP nor INP has an effect on the thawing speed.

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@@ Line 135: / Line 135: @@
-To test the usefulness of the arabinose-inducible promoter I13453 in our 2011 iGEM project, we fused the promoter to a GFP reporter, and assayed the promoter’s activity after addition of different amounts of arabinose. We tested this activity both in a TOP10F’ (figure 1) as well as a MG1655 (figure 2) E.coli strain background. For more information on E.coli strain descriptions, we recommend the following web site: http://openwetware.org/wiki/E._coli_genotypes.
+To test the usefulness of the arabinose-inducible promoter I13453 in our 2011 iGEM project, we fused the promoter to a GFP reporter, and assayed the promoter’s activity after addition of different amounts of arabinose. We tested this activity both in a TOP10F’ (figure 1) as well as a MG1655 (figure 2) ''E.coli'' strain background. For more information on ''E.coli'' strain descriptions, we recommend the following web site: http://openwetware.org/wiki/E._coli_genotypes.
 As can be seen in figure 1, addition of arabinose had only minor, if any, effect on the growth of TOP10F’ cells, most likely because these cells cannot metabolize the sugar. However, to our surprise, we did not observe an arabinose-induced increase of fluorescence compared to the control without arabinose for the first 6 hours. The observed increase in fluorescence after arabinose addition at the 8 hour time point could not be confirmed when the experiment was repeated.
@@ Line 150: / Line 150: @@
 We fused the constitutive promoter to a GFP reporter, and assayed the promoter’s activity after addition of different amounts of arabinose and IPTG. As such these results can serve as a control for the results obtained for the arabinose-inducible BBa_K584000 and IPTG-inducible BBa_K584002 bricks.
-We tested the activity both in a TOP10F’ (figure 1 & 3) as well as a MG1655 (figure 2 & 4) E.coli strain background. For more information on E.coli strain descriptions, we recommend the following web site: http://openwetware.org/wiki/E._coli_genotypes.
+We tested the activity both in a TOP10F’ (figure 1 & 3) as well as a MG1655 (figure 2 & 4) ''E.coli'' strain background. For more information on ''E.coli'' strain descriptions, we recommend the following web site: http://openwetware.org/wiki/E._coli_genotypes.
 Apart from a small growth defect and somewhat lower fluorescence at the 8 hour time point after the addition of 2% arabinose, the activity of the constitutive promoter in TOP10F’ cells is unaffected by arabinose addition, as seen in Figure1. For the MG1655 cells (Figure 2), however, we see clear effects of adding arabinose at concentrations of 0.2% or 2%, with promoter activities significantly lower than the control condition. This points to interference of high inducer levels with the activity of the promoter and the importance of metabolism of the inducer, as arabinose can be metabolized by MG1655 cells, but not by TOP10F’ cells.
@@ Line 160: / Line 160: @@
-To gain insights into the usefulness of the lactose-inducible promoter I13453 in our 2011 iGEM project, we fused the promoter to a GFP reporter, and assayed the promoter’s activity after addition of different amounts of IPTG. IPTG is a molecular mimic of allolactose, a lactose metabolite that triggers transcription of the lac operon. Unlike lactose, IPTG cannot be metabolized by wild-type E.coli cells, leading to a constitutive high presence of inductor. For this reason, IPTG is often used instead of lactose to induce the lac operon.
+To gain insights into the usefulness of the lactose-inducible promoter I13453 in our 2011 iGEM project, we fused the promoter to a GFP reporter, and assayed the promoter’s activity after addition of different amounts of IPTG. IPTG is a molecular mimic of allolactose, a lactose metabolite that triggers transcription of the lac operon. Unlike lactose, IPTG cannot be metabolized by wild-type ''E.coli'' cells, leading to a constitutive high presence of inductor. For this reason, IPTG is often used instead of lactose to induce the lac operon.
-We tested the activity both in a TOP10F’ (figure 1) as well as a MG1655 (figure 2) E.coli strain background. For more information on E.coli strain descriptions, we recommend the following web site: http://openwetware.org/wiki/E._coli_genotypes.
+We tested the activity both in a TOP10F’ (figure 1) as well as a MG1655 (figure 2) ''E.coli'' strain background. For more information on ''E.coli'' strain descriptions, we recommend the following web site: http://openwetware.org/wiki/E._coli_genotypes.
 Addition of IPTG to TOP10F’ cells results in a minor growth defect, as can be seen in Figure 1A. However, this does not seem to inhibit the induction of the promoter, as Figure 1B clearly demonstrates that IPTG results in a clear induction of fluorescence, while without IPTG no such induction is seen.
-In MG1655 E.coli cells, IPTG at a concentration of 1.5mM leads to a minor growth defect (Figure 2A). Looking at the fluorescence measurements (Figure 2B), it is clear that the promoter already displays high activity in the absence of inducer, and that this activity is not increased by adding IPTG. More still, adding IPTG at concentrations of 1mM or 1.5mM even results in a lower fluorescence, and hence promoter activity, than the situation without IPTG addition. We think that this is due to the fact that we express our construct on a multicopy vector, which may outcompete the inhibitory activity of the LacI repressor under these conditions. In contrast to the MG1655 cells, the TOP10F’ strain contains a high copy LacI repressor, making the investigated promoter activity not leaky. These experiments highlight the importance of checking the strain background for compatibility with a desired system.
+In MG1655 ''E.coli'' cells, IPTG at a concentration of 1.5mM leads to a minor growth defect (Figure 2A). Looking at the fluorescence measurements (Figure 2B), it is clear that the promoter already displays high activity in the absence of inducer, and that this activity is not increased by adding IPTG. More still, adding IPTG at concentrations of 1mM or 1.5mM even results in a lower fluorescence, and hence promoter activity, than the situation without IPTG addition. We think that this is due to the fact that we express our construct on a multicopy vector, which may outcompete the inhibitory activity of the LacI repressor under these conditions. In contrast to the MG1655 cells, the TOP10F’ strain contains a high copy LacI repressor, making the investigated promoter activity not leaky. These experiments highlight the importance of checking the strain background for compatibility with a desired system.
+''
 The investigated promoter, pLac-Lux (I13453), should also be repressed by the luxR repressor bound to the corepressor CO6HSL. Due to time limitations, we could not investigate this aspect of the promoter.
 As an additional control, we checked the activity of a constitutive promoter under the same conditions as described here. For results on these experiments, check out our BBa_K584001 page.
@@ Line 175: / Line 175: @@
-To test the usefulness of the cold shock-inducible promoter J45503 in our 2011 iGEM project, we fused the promoter to a GFP reporter, and assayed the promoter’s activity after a temperature shift from 37°C to 25°C or 4°C. We tested this activity both in a TOP10F’ (figure 1) as well as a MG1655 (figure 2) E.coli strain background. For more information on E.coli strain descriptions, we recommend the following web site: http://openwetware.org/wiki/E._coli_genotypes.
+To test the usefulness of the cold shock-inducible promoter J45503 in our 2011 iGEM project, we fused the promoter to a GFP reporter, and assayed the promoter’s activity after a temperature shift from 37°C to 25°C or 4°C. We tested this activity both in a TOP10F’ (figure 1) as well as a MG1655 (figure 2) ''E.coli'' strain background. For more information on ''E.coli'' strain descriptions, we recommend the following web site: http://openwetware.org/wiki/E._coli_genotypes.
 We can see clearly that transferring cells (both TOP10F’ and MG1655) to lower temperatures (4°C and even 25°C) results in a growth arrest between the 1 and 4 hour time points of our experiment (Figures 1A and 2A). We see that promoter activity is induced when cells are transferred to 25°C and even when they are put in and ice bath (4°C) (Figures 1B and 2B). Unfortunately, however, cells that are kept at 37°C also display an increase in promoter activity, indicating leakiness in the system.