Part:BBa_K2984008

PsaD Promoter A1-B1; High-level expression of genes

This part is a part of the Chlamy-HUB-Collection. For the effective and regulated expression of genes a promoter is required. The promoter is located at the 5’ region of the sense strand of the DNA and provides a binding site for the RNA-polymerase and the transcription factors. In the nucleus of the green alga Chlamydomonas reinhardtii a gene encoding a chloroplast protein - PsaD - is located. Fischer and Rochaix (2001) showed that the ORF of this gene does not contain any introns and that the promoter drives a strong constitutive expression of the gene. Using the identified sequence of the promoter a vector for the high-level expression of endogenous and exogenous genes was created (Fischer and Rochaix, 2001).

This part was designed to be used with the MoClo standard and has A1-B1 overhangs.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 18
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

Usage and Biology

For the effective and regulated expression of genes a promoter is required. The promoter is located at the 5’ region of the sense strand of the DNA and provides a binding site for the RNA-polymerase and transcription factors. In the nucleus of the green alga Chlamydomonas reinhardtii a gene encoding a chloroplast protein - PsaD - is located. Fischer and Rochaix (2001) showed that the ORF of this gene does not contain any introns and that the promoter drives a strong constitutive expression of the gene. Using the identified sequence of the promoter a vector for the high-level expression of endogenous and exogenous genes was created (Fischer and Rochaix, 2001).

Characterization

This part was characterized as part of the composite part BBa_K2984019.

Fig.1 - Cloning scheme of L0-PsaD_A1-B1 into E. coli. A PsaD A1-B1 PCR product on agarose gel. B + C Successful transformation into E. coli. D + E Sequencing results of L0-PsaD_A1-B1.

For the characterization of the PsaD promoter, we wanted to see if the part is influenced by light. Therefore, we prepared three different sets of algae 3 days in advance. The first two included C.reinhardtii algae which expressed this composite part. the third one inlcuded our wt algea strain UVM 4. The first and the second were grown under synchronous 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 shaken 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 parameters were used, as for the measurements above. We measured every 20 minutes for approximately 20 hours. Covering the transition from subjective day to night. During the phase of subjective night the plate remained 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 measurements 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 third set is referred to as YFP-wt . Figure 1 is showing the result for the YFP-sync time resolved measurement. The blue plot represents the flourescence intensity while the red line shows the course of the optical density. The grey backround indicates subjective night. It can be seen that the flourescence 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).

Fig. 2 - Time resolved measurement of fluorescence intensity (blue) and optical density (red) of mVenus expressing Chlamydomonas . The algea were grown for three days and then measured for one day under snychronous light conditions. the Grey background indicates the shift to the light phase.

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, as can be seen on Fig. 2. Hereby we aimed to verify that the effect is caused by the light induction and not by syncronization of the algae.

Fig. 3 - Time resolved measurement of fluorescence intensity (blue) and optical density (red) of wild type Chlamydomonas strain UVM 4. The algea were grown for three days in dark and then measured for one day under snychronous light conditions. the Grey background indicates the shift to the light phase.

Compared to the fluorescence intensity (FL) of YFP-sync, the same rise is observable with begin of the light phase (figure 2). In contrast to figure 2 the optical desity (OD) of the YFP-dark set shows a short rise in value, follwed by a decrease shortly after. The fluctuation of the optical density does not seem to correlate with the fluorescence intensity. Even though the rise of OD and FL appear at the same time, the FL remains higher. As we described above Chlamydomonas reinhardtii shows a strong auto-flourescence. To assess the impact of the autofluorescence we measured our wildtype strain UVM 4 as well. The result is depicted in figure 3:

Fig. 4 - Time resolved measurement of fluorescence intensity (blue) and optical density (red) of wild type Chlamydomonas strain UVM 4. The algea were grown for three days and then measured for one day under snychronous light conditions. the Grey background indicates the shift to the light phase.

The wildtype FL only changes slightly during the time of measurement. With beginning of the lightphase a small dip can be observed. Which seems to be corresponds do the rise of the OD. It is notable that the OD for of the wildtype meaurement as well as the YFP-dark measurement (figure 2) show the same type fluctuation at the beginning of the light-phase. We are uncertain how to interpret these fluctuations and think they might be artifacts.

These measurements seem to confirm that the PsaD promoter is light inducible.

References

1. Fischer, N., & Rochaix, J. D. (2001). The flanking regions of PsaD drive efficient gene expression in the nucleus of the green alga Chlamydomonas reinhardtii. Molecular Genetics and Genomics, 265(5), 888-894.