Difference between revisions of "Part:BBa K2009430"

Latest revision as of 09:08, 19 October 2016

sfGFP1-10

Squence And Features

Introduction

sfGFP1-10——PSB1A3 length: 642bp
Derived from: PCR from Part:BBa_I746916，Cambridge 2008

sfGFP1-10——PSB1A3 is an expression plasmid which insert sfGFP1-10 into PSB1A3.

PSB1A3 is a high copy number plasmid carrying ampicillin resistance. The replication origin is aPUC19-derived pMB1.

SfGFP1-10 is a part of GFP(from 1bp to 214bp), GFP has been mutated to improve its solubility and self-associating activity. When it express, it will emit green fluorescence slightly under the fluorescence microscope.

We try to find anideal protein tag to be work both invivo and invitro and it can provide a sensitive measurable signal which don’t need external chemical reagents or substrates. Finally we find away to accomplish this goal—— dividing GFP into sfGFP1-10and sfGFP11. Either the sfGFP1-10 or sfGFP11 will emit green fluorescence slightly under the fluorescence microscope. However, when sfGFP1-10 and sfGFP11 express insame cell, they will interact each other and emit more intense fluorescence than each of them. The split GFP system is simple and does not change fusion protein solubility.

Primers for these biobrick vectors can be found in part:
BBa_G00100 (aka VF2)
BBa_G00101 (akaVR)

The split GFP system has many practical applications. Obtaining soluble, well-folded recombinant proteins for downstream applications requires screening large numbers of protein variants (mutants,fragments, fusion tags, folding partners) and testing many expression or refolding conditions.(Ste´phanie Cabantous, Thomas C Terwilliger & Geoffrey S Waldo,2005)

Part Sequence

Assume Protein Structure

We used Phyre2 to get the assume structure:

(by PyMOL)

Citation:

The Phyre2 web portal for protein modeling, prediction and analysis

Kelley LA et al.

Nature Protocols 10, 845-858 (2015).

Results:

This is the picture which shows Escherichia coli (E.coli) containing the plasmid of sfGFP1-10 observed with excitation light.
This is the picture which shows E.coli containing the sfGFP11 plasmid and sfGFP1-10 plasmid observed with excitation light.
If you want to see all pictures, please go to our notebook.
experimental data:

The ideal results are the spilt GFP experiment in E-coli to verify the split GFP can function as our expectation, we transform the plamids containing sfGFP1-10 and the plasmids containing sfGFP11 respectively in BL21, cultivating the bacteria at 37°C and shacking at 250 rpm/min overnight. We use fluorescence microscope to observe bacteria under 100X objective lens. From these fluorescent images, we find that fluorescent intensity of sfGFP1-10 is stronger than fluorescent intensity of sfGFP11 and both of them are weak. It corresponds with our expectation that either of separate part of sfGFP have background expression and sfGFP1-10, which is longer, may be brighter after excitation.

However, when the PSB1C3 carrying the part of sfGFP11 and the PSB1A3 carrying the part of sfGFP1-10 ware expressed together in E.Coli, it doesn't present stronger fluorescence intensity than either plasmid is expressed in E.Coli respectively. There are a few reasons can explain it. Firstly, different metabolic stress of two plamids causes indistinct results. PSB1C3 carrying sfGFP11, whose gene length is shorter, may have higher expression level than PSB1A3 carry sfGFP1-10. Secondly, there is obvious fluorescence quenching after excitation. Therefore, results of fluorescent images and fluorescent images appear that co-expression of sfGFP1-10 and sfGFP11 has weaker fluorescence intensity. Thirdly, because sfGFP1-10 and sfGFP11 don’t express in E.Coli at 1:1 ratio, the collision probability may be lower than our expectation.

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.