Difference between revisions of "Part:BBa K2909000"

Revision as of 13:30, 6 October 2019

HiBiT-B2 MoClo C. reinhardtii

Sequence and Features

Introduction

1- Biological background

HiBiT Tag developed by Promega to allow for a quick method of protein quantification by luminescence (https://www.promega.com/resources/pubhub/features/hibit-a-tiny-tag-for-antibody-free-endogenous-protein-detection/).
This part is standardized in the Phytobrick MoClo standard for Chlamydomonas reinhardtii.
This tag is flanked on both side by specific fusion sites and BbsI sites to allow its integration into a level 0 plasmid of the C. reinhardtii MoClo Kit.
This tag is designed to be integrated at the B2 position (N-terminal tag).

2- Usage in iGEM projects

Bio(oil)gical Factory (iGEM Sorbonne Université 2019)

Characterization

To Characterize this part along with its C-terminal counterpart (BBa_K2909001), we constructed 5 reporter constructs:
pCMM-1 : ParoR_HiBiT-CloverGFP (BBa_K2909007)= test construct for BBa_K2909000
pCMM-2 : ParoR_CloverGFP-HiBiT (BBa_K2909008) = test construct for BBa_K2909001
pCMM-3 : ParoR_CloverGFP (BBa_K2909004) = negative control construct for the luminescence signal
pCMM-4 : ParoR_NanoLuc-CloverGFP (BBa_K2909005) = positive control construct for the luminescence signal using a N-terminal tag
pCMM-5 : ParoR_CloverGFP-NanoLuc (BBa_K2909006)= positive control construct for the luminescence signal

References

1. Schwinn, M. K. et al. CRISPR-Mediated Tagging of Endogenous Proteins with a Luminescent Peptide. ACS Chem. Biol. 13, 467–474 (2018).
2. Crozet, P. et al. Birth of a Photosynthetic Chassis: A MoClo Toolkit Enabling Synthetic Biology in the Microalga Chlamydomonas reinhardtii. ACS Synth. Biol. 7, 2074–2086 (2018).
3. 1.Weber, E., Engler, C., Gruetzner, R., Werner, S. & Marillonnet, S. A Modular Cloning System for Standardized Assembly of Multigene Constructs. PLoS ONE 6, e16765 (2011).