Overview
In this year’s project, we used both the existing basic parts, including GAL4, VP64, KRAB, 9×UAS, etc., and parts of our own independent design, namely Degron. In the past few years, several teams have constructed and validated composite parts, GAL4-VP64 and GAL4-KRAB. However, they only focused on the degree of activation of gene expression by GAL4-VP64 and the degree of inhibition of gene expression by GAL4-KRAB in different systems, and did not address the effect of these two transcription factors acting together on the same part. In our design, these two parts are placed in the same biosystem to further explore the co-regulatory effects of these two parts on genes. On the other hand, we designed the Degron part independently.
All of these may be helpful to other teams, and we hope it will make contributions to the iGEM community.
Our first contribution
1. Our first contribution was to construct two plasmids, pcDNA3.1(+)-3×HA-GAL4-VP64-NLS and pGL4.35-3×HA-9×GAL4UAS-KRAB-NLS, to achieve rhythmic expression of GnRH.
We obtained new plasmids that could be used by recombining CMV, GAL4, VP64, KRAB, 9×UAS and other parts with existing plasmid vectors. GAL4 is a protein that can find and bind UAS (upstream activation sequence). VP64 is a transcription factor that, when used in combination with GAL4, can activate UAS and initiate the expression of downstream genes. KRAB is also a transcription factor that, when combined with GAL4, can also bind to UAS, but represses downstream gene expression. In our project, we used GAL4-VP64 to initiate GAL4-KRAB expression, while GAL4-KRAB can repress its own expression. This constitutes a complete negative feedback regulatory loop that can render rhythmic expression of GnRH with an upstream regulatory sequence of 9×UAS.
Figure : Luciferase achieves rhythmic secretion. When GAL4-VP64=GAL4-KRAB=100ng (Luciferase=800ng, pcDNA3.1 empty vector=1400ng in the transfection system), Luciferase cycling period T=33.12h.
The idea was validated through our experimental investigations. GAL4-VP64 and GAL4-KRAB can indeed be engineered to establish a negative feedback regulatory loop, inducing rhythmic expression of GnRH. The regulation of the GnRH period could be achieved through three approaches: (1) controlling the mass of GAL4-VP64 and GAL4-KRAB plasmids; (2) adjusting the mass ratio of GAL4-VP64 to GAL4-KRAB plasmids; (3) utilizing the Degron sequence.
Our second contribution
2. Our second contribution involves the construction of the Degron fragment and its utilization in generating two additional recombinant plasmids, pcDNA3.1(+)-3×HA-GAL4-VP64-Degron-NLS and pGL4.35-3×HA-9×GAL4UAS-KRAB-Degron-NLS.
Degron is a short peptide consisting of 41 amino acids, which enables specific binding of protein substrates by the E3 ubiquitin ligase within the ubiquitin-proteasome system. This leads to substrate ubiquitination and subsequent degradation, thereby reducing protein half-life. By incorporating DNA sequences corresponding to Degron into existing plasmids, we successfully shortened the half-lives of GAL4-VP64 and GAL4-KRAB proteins. Based on this, we further reduced GnRH expression period by introducing these two recombinant plasmids containing Degron into the aforementioned negative feedback regulatory loop.
Figure : Biofluorescence intensity when GAL4-VP64:GAL4-KRAB=1:1
Figure : Biofluorescence intensity when GAL4-VP64-Degron:GAL4-KRAB-Degron=1:1
