Difference between revisions of "Part:BBa K3927001"
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===Description===
 
===Description===
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https://static.igem.org/mediawiki/parts/thumb/4/47/T--NUS_Singapore--C120-CYC_schematic.png/800px-T--NUS_Singapore--C120-CYC_schematic.png
  
 
===Usage===
 
===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===
 
===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===
  
 +
<b> Characterization of expression from an episomal plasmid </b>
 +
 +
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).
 +
 +
[[File:T--NUS_Singapore--Induction_of_pC120-mKO-EL-U.png|700px|thumb|center|Figure 2: BY4741 wildtype compared to BY4741 containing pC120-promoter controlling mKO as well as constitutive expression of NLS-VP16-EL222.]]
 +
 +
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.
 +
 +
https://static.igem.org/mediawiki/parts/thumb/1/10/T--NUS_Singapore--Expression_of_C120-mKO-EL222-U_overtime.png/800px-T--NUS_Singapore--Expression_of_C120-mKO-EL222-U_overtime.png
 +
 +
<i> Figure 3: Comparison of mKO expression over time in complete darkness, complete blue light or 50% blue light, implemented using alternating cycles of 30 minutes blue light and 30 minutes darkness. </i>
 +
 +
<b> Characterization of expression from genome integrated cassette </b>
 +
 +
https://static.igem.org/mediawiki/parts/thumb/e/ed/T--NUS_Singapore--Improvement_of_blue_light_system.png/800px-T--NUS_Singapore--Improvement_of_blue_light_system.png
 +
 +
<i>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.</i>
 +
 +
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===
 
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
 
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
 
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
  

Latest revision as of 03:48, 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


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal XbaI site found at 104
    Illegal SpeI site found at 79
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 86
    Illegal SpeI site found at 79
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal XbaI site found at 104
    Illegal SpeI site found at 79
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal XbaI site found at 104
    Illegal SpeI site found at 79
  • 1000
    COMPATIBLE WITH RFC[1000]

Description

800px-T--NUS_Singapore--C120-CYC_schematic.png

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: BY4741 wildtype compared to BY4741 containing pC120-promoter controlling mKO as well as constitutive expression of NLS-VP16-EL222.

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.

800px-T--NUS_Singapore--Expression_of_C120-mKO-EL222-U_overtime.png

Figure 3: Comparison of mKO expression over time in complete darkness, complete blue light or 50% blue light, implemented using alternating cycles of 30 minutes blue light and 30 minutes darkness.

Characterization of expression from genome integrated cassette

800px-T--NUS_Singapore--Improvement_of_blue_light_system.png

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