Difference between revisions of "Part:BBa K2009701"

Revision as of 08:56, 19 October 2016

SUP35+sfGFP11

Sequence And Features

introduction

Sup35——PSB1C3 length: 783bp
Derived from: PCR from the plasmid provided from Dong Men

We construted this part as a component of our pro priontein system.

This composite part is contructed by ligating SUP35NM gene and the gene of sfGFP11(with linker).

The split GFP system is simple and does not change fusion proteinsolubility. Either the sfGFP1-10 or sfGFP11 will emit green fluorescence slightly under the fluorescencemicroscope. However, when sfGFP1-10 and sfGFP11 express insame cell, they will interact each other and emit more intense fluorescence thaneach of them.

Sup35p is a subunit of the translation termination complex. In nature, it has been proved to have kinds of prionic effect on the stress response, When the conformation is transformed into prionic form, the termination of translation is less effective and thus new longer proteins form (True and Lindquist, 2000; Tyedmers et al., 2008). Research found that if the sequence corresponding to the NM domains of Sup35p is fused to other gene, the protein resulting of this construction acquires the prionic behaviour (Li and Lindquist, 2000). Tyedmers et al. (2000) found that heat shock is a significantly relevant factor to trigger the prionic conformation. Heat shock will induce The cells the prionic phenotype, it is an autocatalytic process and finally, all the protein is found in the prionic conformation. The cells resulting show the phenotype [PSI+]. One of the most possible reason for phenomenon is that HSP104 play an important role in the formation and maintenance of the amyloid fiber (Halfmann et al., 2009). The SUP35NM gene we used is provided by Dong Men, PhD of Wuhan Insititue of Virology of Chinese Academy of Sciences, so that the sequence is not quite the same as the existing sequence in the Parts Registry, for a lot of mutations have been done. The standardization of this gene is did by ourselves by adding the Biobrick prefix and suffix to its ends and doing a site mutation to eliminate the PstI cutting site inside it.

Part Sequence

Results:

The spilt sfGFP experiment in Yeast(S. cerevisiae,W303)

Firstly, we transformed the two kinds of plasmids into the S. cerevisiae W303 to get three types of S. cerevisiae respectively containing sfGFP1-10, PR-sfGFP11 and both. Then we cultivated the cells at 30 ℃ for 24 hours.

(1) Testing the effect of heat shock

We measured fluorescence intensity of two groups of S. cerevisiae cultivated separately at 30,34,38,42 ℃ without GdnHCl. The first group was cultivated for 1 hour and the other one for 2 hours.

Here are original images of the two groups’ fluorescence experiment.

Heat Shock 1h:

<brHeat Shock 2h

Heat Shock 2h:

By using Image Processing method described in the appendix, we got these two diagrams.

As shown in the Figure 1 (2 hours of heat shock) above, the brightness of the bright dots in the photo, which represents the level of sfGFP 1h after heat shock, decreases almost linearly as the temperature increases. According to the linear fitting, the brightness drops about 5 units for the increase of temperature of 1°C, which is about 3% of the level at 30 degrees Celsius. In the modeling part, we predicted a linear decreasing relationship between temperature and the sfGFP output level. So it coincides with our experimental results.
It is noteworthy that high temperature is likely to affect the binding of sfGFP1-10 and sfGFP11, and destabilize the sfGFP complex. So we must exclude the possibility that the result in the figure above can be only attributed to this factor, instead of the aggregation of Sup35. According to Zhang et al, from 30 degrees to 42 degrees, the fluorescence intensity decreases for about 20%, owing to the effect of high temperature. However, in our experiment, it decreases with 35%, which is obvious more than the effect of temperature only. So there must be another mechanism, which should be that aggregation of Sup35 blocks sfGFP11 and make it impossible or at least harder to bind with sfGFP1-10. What’s more, the difference of the decreasing ratio, 15%, is precisely identical to the predicted ratio in our modeling result. Thus we can safely draw the conclusion that the sfGFP level decreases with increasing temperature, due to or at least partly due to the aggregation of Sup35.
As for the Figure 2 (2 hours of heat shock), it shows that there is a huge decrease of fluorescence intensity when temperature increases from 30 ℃ to 34 ℃ while no obvious changes occur when temperature varies from 34 ℃ to 42 ℃. That is not fully in line with expectation. The possible explanation is that heat shock indeed causes the aggregation of Sup35 and relatively higher temperature can enhance the aggregation effect. But there is another important factor you have to notice, which is that when Sup35 is in prion state (non-prion state of Sup35 nearly don’t form aggregation), it can propagate and transform the normal Sup35 protein into its prion state. And 2 hours is so long for the cells to finish the form changing process and aggregation of all the Sup35, regardless of the little differences in the temperature of heat shock. In conclusion, the latter three points in diagram 2 have similar values because the aggregation of Sup35 had come to saturation. Now you might want to ask why group 1 doesn’t show this contradiction. The reason might be that 1 hour is not sufficient for process of injection and aggregation to finish and during that time temperature has a dominant effect on the aggregation of Sup35.
In a word, from the temperature experiments above, we provide valid evidence which showed that our system could work effectively!

(2) Testing the effect of GdnHCl

We measured fluorescence intensity of S. cerevisiae cultivated at 42 ℃ in 0.25 mM, 0.50 mM, 3 mM, 5 mM GdnHCl for 4 hours.


According to statistics analysis, with the increase of concentration of GdnHCl, fluorescence intensity increase in the first stage, and then decrease. In the first stage, fluorescence became brighter because Sup35 disaggregated and our kill switch turned on again. In the latter stage, the fluorescence intensity decreases which was opposite to our modeling results. We assumed that GdnHCl, which possesses high electric charge, may lead to the misfolding of split sfGFP. It is likely to disturb the two fragments assembling and reconstituting. Thus, GdnHCl is a suitable curing reagent for Sup35 in S. cerevisiae within limited concentration range.