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Submitted Parts

Submitted Biobricks
Fig 1. Our submitted BioBricks consist of three parts: the Degron, the Gal4-VP16 Transcriptional activator, and Gal4-Degron-VP16 complex.

Degron (BBa_K1408000)

The Degron is an unstable protein domain that, when fused to a protein, acts as a source of instability. The presence of the Degron leads to degradation of the protein by the cell via ubiquitination. The Degron was obtained from Ben Jester, from the Fields Lab (UW Genome Sciences). This is a new part we are submitting to the registry.

Gal4-VP16 Transcriptional Activator ( BBa_K1408001 )

This part consists of our Gal4-VP16 fusion protein, which is a transcriptional activator. Gal4 protein binds to a Gal1 promoter, while VP16 recruits transcription machinery, promoting transcription to anything under the Gal1 promoter. The two must be co-localized to work as a transcriptional activator.

This transcrptional activator has been previously submitted to the registry (BBa_K1179014 submitted by the iGEM 2013 MIT team). However, the part was incomplete compared to ours due to the presence of an illegal restriction site that cut the VP16 gene, rendering it useless. Also, this part had no user data related to it. In order to make the complete part available, together with experimental data related to the part, we decided to "re-submit" this part to the registry.

Gal4-Degron-VP16 ( BBa_K1408002)

This part is a combination of our previous two parts submitted to the registry. By utilizing this system, the relative stabilities of proteins can be compared. A sequence for a protein inserted between the Degron and VP16 will produce a fusion protein which acts as a transcriptional activator for anything under a Gal1 promoter. If the protein inserted is unstable it will be degraded by the cell and the Gal4-VP16 transcriptional activator will no longer function. If a quantifiable marker protein such as Green Fluorescent Protein (GFP) is under Gal1 then the level of GFP output of the cell is related to the stability of the inserted protein.

Our collection of three parts consists of a degron, the test plasmid containing the GAL4-VP16 transactivator, and a construct used in evolving our protein for higher GFP.With those two parts alone, BBa_K1408001 and BBa_K1408002, users can use BioBrick cloning or PCR to generate ALL 5 test constructs: Deg0, Deg1, Deg2, Deg3, and Deg4. We chose to submit these parts separately to allow future users the utmost freedom in designing a specific test construct for their specific protein of interest.