Coding

Part:BBa_K1470002

Designed by: Pascal Sartor   Group: iGEM14_Freiburg   (2014-10-02)

Galactose-induced gene 4 DNA binding domain (Gal4DBD)

Usage and Biology

This protein from Saccharomyces cerevisiae is a positive transcriptional regulator for the gene expression of the galactose-induced genes such as GAL1, GAL2, GAL7, GAL10, and MEL1 which are important for galactose import and conversion to glucose [1]. It recognizes a 17 base pair sequence (5'-CGGN11CCG-3') in the upstream activating sequence (Gal4UAS) of these genes. [2] Gal4DBD is used to investigate gene expressions in several organisms (bacteria, plants, fruit flyes) [3]. -The protein is controlled by a strong promotor. Binding to Gal4UAS leads to the expression and detection of the corresponding gene, GFP for example.

To demonstrate the functionality of Gal4DBD, experiments with the blue light inducible gene expression system were used. The blue light inducible gene expression system can not work without Gal4DBD because the Gal4DBD binds to the Gal4UAS and marks the connection between the LOV2 domain and the Gal4UAS. This sequence positions the system close to the promoter of the reporter gene, thereby ensuring functionality of the system. For a better understanding, read in the following description.

Figure 1: Principle of the blue light inducible expression system based on the LOV2 domain of phototropin from the common oat. Avena sativa.


The blue light expression system used for the AcCELLarator capitalizes on the second LOV (Light-Oxygen-Voltage) domain of the protein phototropin of Avena sativa (AsLOV2). LOV domains are small photosensory peptides with up to 125 residues and are used by many higher plants, microalgae, fungi and bacteria to sense environmental conditions. LOV domains, such as the AsLOV2 domain, have been successfully employed in several designs for optogenetic tools. AsLOV2 is N- and C-terminally flanked by α helices, referred to as the Aα and Jα Helix, respectively. In addition to the LOV2 domain, there are several other parts necessary for light induced expression of target genes. They can be separated into two main modules: One includes the previously mentioned LOV2 domain that is fused to a Gal4-DNA binding domain (Gal4DBD). This part is constitutively bound to a specific DNA sequence, the Gal4-upstream activator sequence (Gal4UAS) nearby the promotor region of a target gene. The second part consists of an engineered PDZ domain (ePDZ) that is fused to a VP16 domain. The VP16 domain can act as a transcriptional activator which recruits DNA polymerase to the gene of interest. The interaction between ePDZ and the Jα helix of LOV2 is the key process of the system: While in the dark, the Jα chain is not exposed, therefore, the ePDZ-VP16 domain cannot be recruited, and the gene of interest will not be transcribed. Upon illumination, the Jα chain of the LOV2-domain becomes accessible, enabling the second part of the light system, ePDZ fused to VP16, to bind to the Jα chain. VP16 recruits DNA polymerase, thereby leading to transcription of the gene of interest.

Figure 2: Plasmid map of pKM292. The Gal4DBD fused to a LOV domain. Together with pKM297 used for blue light induced gene expression. Plasmid list

Figure 3: Plasmid map of pKM297. The ePDZ fused to the activater domain VP16. Together with pKM292 used for blue light induced gene expression. Plasmid list

Figure 4: Plasmid map of pKM084. The reporter plasmid pKM084 with SEAP is used together with pKM292 and pKM297 for blue light induced gene expression. Plasmid list

We obtained pKM292, pKM297 and pKM084 from Konrad Müller, AG Weber of the University of Freiburg. Gal4DBD, ePDZ and SEAP were stardardized and send in the shipping backbone pSB1C3 by us.



The blue light system is effective in expressing the reporter SEAP which was introduced into the cells before illumination by transfections. As we know that the duration of illuminating the system with blue light (452 nm) is critical for its efficiency, we tested various time intervals for illumination. Our results indicate that five hours of illumination lead to the highest level of SEAP expression (Fig. 4). Another important finding was the specificity of the blue light system. In the dark controls, we found almost no activation of the SEAP reporter, leading to a very low background level in our system. The results of the blue light systems were measured by SEAP expression:

Figure 5: Efficiency of the blue light system using different time intervals of illumination. To test the efficiency of the light system, SEAP expression after illumination was determined. The SEAP assay was performed 24 hours after light exposure. Cells were incubated with blue light for 1 hour, 2.5 hours and 5 hours.

More information

Figure 6: Kinetics of the blue light induced receptor. HEK-293T cells were transfected with the blue light system (PKM292 and PKM297) and the light induced receptor (p14ls_003, mCherry linked receptor). Pictures were taken after 12h, 15h, 18h and 24h.

More information


Contribution
Group: Team Tsinghua 2016.
Author: Tianyang Mao.
Summary: Section below is the contribution Team Tsinghua made to previous parts . We characterized three parts, one of which belongs to this page (http://parts.igem.org/Part:BBa_K1323002, http://parts.igem.org/Part:BBa_K1493504 and http://parts.igem.org/Part:BBa_K1470002) by transforming the part into E. coli and validated its sequence using enzymatic digestion as well as sequencing. We functionally improved these parts by fusing three parts together (dCas9 from BBa_K1323002, GFP from BBa_K1493504, and Gal4BD from BBa_K1470002). In a sentence, the nuclear localization of dCas9 protein can be well visualized. More controlled experiments suggest we successfully repurposed CRISPR/Cas9 for transcriptional control of an exogenous suicidal system contingent upon the fidelity of canonical mRNA sequences. For detailed documentation, please refer to the part page http://parts.igem.org/Part:BBa_K1923011.


Imporve
Group: Shenzhen_SFLS
Author: Runqi zhu
Summary: The contribution team SFLS made to this part is as follow. Our part BBa_K2200007 (http://parts.igem.org/Part:BBa_K2200007) went a step further to improve this part by adding a part of the transcriptional activation domain of the NF-KB p65 protein fused to the DNA-binding domain of GAL4 protein. GAL4 is used as a TAS (transcriptional amplification strategy system) to enhance the transcription of weak tumor-specific promotors. It is proved by experiments that adding p65 protein to the GAL4 protein can significantly improve the expression of downstream gene.

References

[1] TRAVEN, A., JELICIC, B., SOPTA, M.: Yeast Gal4: a transcriptional paradigm revisited. EMBO Rep. May 2006; 7(5): 496–499.
[2] CAMPBELL, R.N., LEVERENTZ, M.L., RYAN, L.A., REECE, R.J.: Metabolic control of transcription: paradigms and lessons from Saccharomyces cerevisiae. Biochem. J. (2008) 414 (177–187).
[3] BRAND, AH., PERRIMON, N.: Targeted gene expression as a means of altering cell fates and generating dominant phenotypes. Development. 1993 Jun;118(2):401-15.
[4] MUELLER, K., ENGESSER, R., TIMMER, J., ZURBRIGGEN, M. D., Weber W.: Orthogonal Optogenetic Triple-Gene Control in Mammalian Cells. ACS Synth. Biol., Article ASAP



Sequence and Features

Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal XhoI site found at 218
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 137


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