Difference between revisions of "Part:BBa K3927001"

Revision as of 03:31, 18 October 2021

C120-CYC

This part encodes for a truncated CYCp core promoter with C120 sequence replacing the native upstream activating sequence.

Sequence and Features

Description

Usage

NLS-VP16-EL222 was expressed constitutively using the native promoter ACT1p in S.cerevisiae strain BY4741 from an episomal plasmid alongside C120-CYC upstream of the fluorescent protein mKO (Figure 1). BY4741 containing this construct was exposed to blue light as well as darkness for 6 hours, and compared to BY4741 without the construct present (Figure 2).

Design

Blue light induced activation of this promoter is dependent on simultaneous expression of an EL222 based transcription factor containing a nuclear localization sequence, and an activation domain.

Characterization

Characterization of expression from an episomal plasmid

NLS-VP16-EL222 was expressed constitutively using the native promoter ACT1p in S.cerevisiae strain BY4741 from an episomal plasmid alongside C120-CYC upstream of the fluorescent protein mKO(Figure 1). BY4741 containing this construct was exposed to blue light as well as darkness for 6 hours, and compared to BY4741 without the construct present (Figure 2).

Figure 2 demonstrates that in the presence of blue light, mKO expression is increased roughly 3 fold compared to darkness. mKO expression was also measured over time for 6 hours(Figure 3) and a comparison was made for 100% blue light, 100% darkness and 50% blue light duty cycles. While constitutive blue light increased expression over time, and constitutive darkness decreased expression over time, 50% blue light maintained roughly constant expression, demonstrating the ability of this part to modulate dose-dependent expression.

Characterization of expression from genome integrated cassette

Figure 4: Expression of C120 controlled mKO from an episomal plasmid, with NLS-VP16-EL222 expressed from the plasmid compared to NLS-VP16-EL222 expressed from the genome.

An identical expression cassette was integrated into the genome into the LTR YPRCd15, and an experiment was carried out containing the minimal C120-promoter maintained in an episomal plasmid, and overall expression of mKO after 6 hours was measured and compared to the system maintaining NLS-VP16-EL222 episomally(Figure 4). Expression of mKO from the construct expressing NLS-VP16-EL222 from a genomic integration cassette was lower than the construct expressing NLS-VP16-EL222 from an episomal plasmid. According to the model developed by the NUS iGEM 2021 team, this can be explained by an decrease in expression due to a reduction in copy when NLS-VP16-EL222 is expressed from the genome as compared to an episomal plasmid.

References

1. Motta-Mena, L. B., Reade, A., Mallory, M. J., Glantz, S., Weiner, O. D., Lynch, K. W., & Gardner, K. H. (2014). An optogenetic gene expression system with rapid activation and deactivation kinetics. Nature chemical biology, 10(3), 196–202. https://doi.org/10.1038/nchembio.1430

2. Benzinger D, Khammash M. Pulsatile inputs achieve tunable attenuation of gene expression variability and graded multi-gene regulation. Nat Commun. 2018 Aug 30;9(1):3521. doi: 10.1038/s41467-018-05882-2. PMID: 30166548; PMCID: PMC6117348