Difference between revisions of "Part:BBa K3927001"
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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. | 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. | ||
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| + | <b> Characterization of expression from genome integrated cassette </b> | ||
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| + | 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=== | ===References=== | ||
Revision as of 03:23, 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
- 10INCOMPATIBLE WITH RFC[10]Illegal XbaI site found at 104
Illegal SpeI site found at 79 - 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 86
Illegal SpeI site found at 79 - 21COMPATIBLE WITH RFC[21]
- 23INCOMPATIBLE WITH RFC[23]Illegal XbaI site found at 104
Illegal SpeI site found at 79 - 25INCOMPATIBLE WITH RFC[25]Illegal XbaI site found at 104
Illegal SpeI site found at 79 - 1000COMPATIBLE WITH RFC[1000]
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
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