Difference between revisions of "Part:BBa K2984019"

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L1c-Psad-YFP-RbcS2, Expression of YFP through light-inducible promoter

This part is a part of the Chlamy-HUB-Collection . This Level 1 construct is designed for comparison of locus dependent expression through fluorescence intensity measuerments. The Construct consist of the PsaD promoter promoter a coding sequence for the yellow fluorescent protein mVenus and the Rbcs2 Terminator. The promoter is inducible by light.

Sequence and Features

Usage and Biology

This construct is designed for the expression of the yellow fluorescent protein (YFP) <a href="https://parts.igem.org/wiki/index.php?title=Part:BBa_K2984017">mVenus</a> in C. reinhardtii. It is composed of the <a href="https://parts.igem.org/wiki/index.php?title=Part:BBa_K2984008">PsaD promoter</a>, which stems from the photosystem one complex, the <a href="https://parts.igem.org/wiki/index.php?title=Part:BBa_K2984017">mVenus</a> coding sequence and the <a href="https://parts.igem.org/wiki/index.php?title=Part:BBa_K2984018">Rbcs2 Terminator</a>, taken from the Rubisco gene. This construct will make C. reinhardtii glow under a fluorescence microscope.

The YFP mVenus has an excitation peak at 515 nm and an emission peak at 528 nm (Kremers et al. 2006). With this construct we focused on the characterization of the light induction of PsaD. Nevertheless, there are many possible uses for this construct. For example, the construct can be used for the comparison of expression rates in dependency of the locus of transgene insertion.

Characterization

The first qualitative characterization we made was to check the mVenus fluorescence of C. reinhardtii that had been transformed with this construct under a fluorescence microscope.

After seeing fluorescence under the microscope, we proceeded to measure the fluorescence intensity of our clone under a plate reader (Fig. 2). Measuring the fluorescence intensity of YFP in C. reinhardtii can be quite challenging, due to the strong interaction of the algae with light (photo systems, chlorophyll, pigments and light antennae). You can find more information about how to measure this part on the measurements siteof the Humboldt Berlin team 2019.

For the measurement we prepared YFP expressing clones and WT algae, to compare the fluorescence of both probes. This is imperative due to the strong autofluorescence of the algae. Additionally, we made sure to have the same optical density (cell concentration) in both YFP and WT probes. We did a sequential dilution in three steps with the probes and measured the fluorescence intensity for each probe. We excited at 490 nm and measured the emission at 528 nm. We were able to show that the fluorescence intensity of the YFP expressing clones was always above the autofluorescence of the wild type, as can be seen in Fig. 2.

After measuring the fluorescence intensity we also measured the emission spectrum of mVenus in C. reinhardtii by doing an emission scan of both YFP expressing clones and WT algae. By doing a difference spectrum of the fluorescence emission spectra of the mentioned probes, we received the emission spectrum of YFP, as can be seen on Fig. 3.

Psad Characterization - Light induced expression of YFP

As further characterization of the psad promoter, we wanted to see if the part is influence by light. Therefor we prepared three different sets of algae 3 days in advance. The first teo included C.reinhardtii algea which expressed our this composite part. the third one inlcuded our wt algea strain UVM 4. The first and the second were grown under synchronos light conditons, meaning they were exposed to light for 14 hours and to darkness for 10 hours. The second set remained in darkness the hole time. All three were constantly shaked at speed of 110 round per minute while temperature was set to 21,4°C. The algea were grown in a black 96-well plate. After three days of growth we startet a time resolved fluorescence intensity measurement.The same paramters were used as for the measurements above. We measured every 20 minutes for approximately 20 hours. Covering the transition from sunjective day to night. During the phase of subjective night the plate remaind in darkness inside the plate reader, or outside under a light source at subjective day. It is important to shake the plate with algae between m measurement to avoid the aggregation of algae on the bottom of the wells, which ultimately affects the measurement of the YFP.

In the following the first and second set are described as YFP-sync and YFP-dark alluding to the different growth conditions. The thrid set is refferd to as YFP-wt . Figure 4 is showing the result for the YFP-sync time resolved measurement. The blue plot is represents the Flourescense intensity while the red line shows th course of the the optical density. The grey backround indicates the switch from subjective night to day. It can be seen that the flourescense indcates shows a clear rise with the beginning of the day phase. The optical density however seems to show no reaction to the change of light intensity (as expected).

One argument might be, that the flourescence signal (and therfore the mVenus expression) rises due to synchronisation of the cells. To verify the effect of light onto the expression of the mVenus we prepared the YFP-dark set:

Compared to the fluorescence intensity (FL) of YFP-sync, the same rise is observable with beginn of the light phase (figure 5). In contrast to figure 5 the optical desity (OD) of the YFP-dark set shows a short rise in value, follwed by a decent shortly after. The fluctuation of the optical density does not seem to corelate with the fluoprescence intensity. Though the rise of OD and FL appear at the same time, the FL remains higher.

References

1. Kremers, G. J., Goedhart, J., van Munster, E. B., & Gadella, T. W. (2006). Cyan and yellow super fluorescent proteins with improved brightness, protein folding, and FRET Förster radius. Biochemistry, 45(21), 6570-6580.