IGEM:IMPERIAL/2007/Experimental Design/Phase1/Results 2.1

In vitro Testing of pTet-GFP and pT7-GFP Constructs

Aims

To determine if the following constructs work in vitro:

• pTet-GFP
• pT7-GFP

To test the operating range of the constructs at 10°C, 37°C and 45°C over a staggered 24 hour period.

To identify problems in experimental methodology.

The testing was comprised of several tests:

• pTet and pT7 in vitro - Tested 21-08-2007 to 23-08-2007
• pTet in vitro at 10°C, 37°C and 45°C - Tested 21-08-2007

Materials and Methods

Refer to protocols page.

Results

pTet-GFP (100ng/μl)

Test: 21-08-2007

In vitro testing at 37°C

Fig.1: GFP Expression of pTet-GFP in vitro

The pTet-GFP contruct was tested in vitro using commercial S30 Cell Extract at 37 °C. As shown in Fig.1, pTet showed a marked increase in the first hour before levelling, reaching a slow but steady increase throughout a period of 4 hours.

Test: 21-08-2007 to Test: 23-08-2007

pTet-GFP in vitro GFP expression at 37°C

Fig.2: GFP Expression of pTet-GFP in vitro at 37°C over 56 hours

Fig.2 shows the incomplete curve of fluorescence levels over a staggered time period. As expected, the fluorescence levels of the pTet construct increase marked during the first few hours. This fluorescence level was higher, albeit reducing, over the middle portion of the graph, where it decreased to less than half the maximum level measured at the end of the time course. Interestingly, the supposed positive control of diluted GFP solution showed similar patterns in terms of fluorescence levels.

Test: 22-08-2007

pTet-GFP in vitro expression at 10°C

Fig.3: GFP Expression of pTet-GFP in vitro at 10°C over 4 hours

There appears to be no significant increase in fluorescent levels of the pTet construct over the measured time course. Also shown in Fig.3 is a decrease in fluorescence levels across all samples and controls. Experiment was not continued over required time course due to lab and safety cosntraints.

Test: 22-08-2007

pTet-GFP in vitro GFP expression at 45°C

Fig.4: GFP Expression of pTet-GFP in vitro at 45°C over 4 hours

There appears to be no significant increase in fluorescent levels of the pTet construct over the measured time course. Also shown in Fig.4 is a decrease in fluorescence levels across all samples and controls. Experiment was not continued over required time course due to lab and safety cosntraints.

pTet-GFP (100ng/μl)

Test: 21-08-2007 to 22-08-2007

In vitro testing at 37°C

Fig.5: GFP Expression of pTet-GFP in vitro

There appears to be no significant increase in fluorescent levels of the pT7 construct over the measured time course. Also shown in Fig.5 is a decrease in fluorescence levels across all samples and controls. Experiment was not continued over required time course due to lab and safety cosntraints.

pT7-GFP in vitro GFP expression at 37°C

Fig.6: GFP Expression of pT7-GFP in vitro at 37°C over 29 hours

The plate containing the samples was stored in a 37oC incubator overnight. It was re-tested the next morning to see whether GFP had been expressed,22 hours after of induction. The results were joined with the initial testing done over the first 4 hours of induction and are shown below. Fig.6 shows that fluorescence levels of the sample increased minimally over the 2 days. This pattern of increase is also apparent in the negative control. Overall fluorescence levels also decreased in the positive control.

Controls:

• Positive control - diluted GFP solution of equal volume
• Negative control - S30 cell extract of equal volume

Complete set of results and raw data

Discussion

pTet-GFP

In vitro testing at 37°C

Fig.1 indicates that there was a fair amount of expression of GFP with the pTet-GFP construct, leading to an increase in fluoresence over time. Although the level of expression is starkly reduced as compared to those in vivo, the construct is shown to be consistently working well in vitro.

In vitro GFP expression at 10°C, 37°C and 45°C over a staggered time period

Due to lab and safety constraints, a staggered time period cannot be obtained for GFP expression at 10°C 45°C temperatures. The results from both these experiments (Fig.3,4) also do not indicate much expression at these temperatures. In comparison, it was observed that the fluorescence levels of the positive control at 45°C decreased more quickly than that in the 10°C one. This may be due to protein instability at higher temperatures. Nevertheless, these results are not conclusive of whether the decay is proportionate or exponential.

Similar patterns of fluorescence level curves from samples and controls in Fig.2 suggests that there may be some major issues in our experimental methodology that require further investigation. It is postulated that evaporation could play a major role in the inconsistency that we have observed. Reducing evaporation to ensure consistent volume of the reaction mixture is ideal to ensure a singular variable in the experiment. Other factors also include the different batches of S30 cell extracts used, the different method of maintaining temperature (water baths and incubators), DNA concentration, and variability in the fluorometer instrument.

Although the initial data corresponds well with previous 37°C in vivo experiments, the absence of a contiguous time course means that it is not feasible to extrapolate the data given the vast difference in fluorescence levels over the three stages.

pT7-GFP

In vitro testing at 37°C

Likewise with the in vivo tests, the pT7-GFP construct did not give the expected increase in fluorescence levels over time. Although this can be attributed to the further reduction in fluorescent signals due to lower expression in the in vitro chassis, it seems more likely that the problem lies more with the construct than other factors.

In vitro GFP expression at 37°C over a staggered time period

Fig.5 indicates a minimal increase in fluorescence levels over a 29 hour period, suggesting that not enough GFP has been expressed for a significant change in fluorescent readings. Although the uncertainty of the results as described above is applicable to this experiment as such, just by judging from the relative total fluorescence alone would indicate that pTet-GFP construct is a more viable option to our design.

Conclusion

• pTet-GFP construct consistently gives higher levels of fluorescence than pT7-GFP construct and should be preferred.
• pTet-GFP construct works in vitro.
• Major factors in our experimental methodology (eg. DNA concentration, evaporation, staggered) might account for the non-correlative fluorescence levels, as with the observed pattern of all curves across the time periods.