Difference between revisions of "Part:BBa K3570021:Experience"

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how you used this part and how it worked out.
 
how you used this part and how it worked out.
  
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===Applications of BBa_K3570021===
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===Applications===
  
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===Description===
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===Applied use of BBa_K3570021 by the Team NUS Singapore 2021===
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NLS-VP16-EL222 is a blue light-sensitive transcription factor. EL222 contains an LOV domain that forms homodimers in the presence of blue light and induces binding to a C120 consensus DNA motif(TAGCCTTTAGTCCATG). This part has been developed for use in the yeast S.cerevisiae and contains a nuclear localization sequence and VP16 activation factor functional in S.cerevisiae.
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===Usage===
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As this part has not been successfully cloned and tested yet, Team NUS Singapore 2021 has performed basic assembly and characterization of this construct as part of our contribution to the registry. We have also made use of this part for our optogenetic project for iGEM 2021, and more detailed descriptions of how our team has utilized this part can be found at our team wiki page: '''https://2021.igem.org/Team:NUS_Singapore'''
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NLS-VP16-EL222 can be expressed in S.cerevisiae as a trans-regulatory factor for a promoter containing a C120 motif in the upstream activation position relative to the core promoter. In blue light, NLS-VP16-EL222 dimerizes and binds to the C120 motif, and a conjugated VP16 domain will promote transcription of downstream elements.
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===Characterization of expression from an episomal plasmid===
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'''Characterization of expression NLS-VP16-EL222 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 a minimal C120 containing promoter 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)
 
NLS-VP16-EL222 was expressed constitutively using the native promoter ACT1p in S.cerevisiae strain BY4741 from an episomal plasmid alongside a minimal C120 containing promoter 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)
  
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https://static.igem.org/mediawiki/parts/3/3d/T--NUS_Singapore--Construct_tested_with_NLS-VP16-EL222_expressed_from_an_episomal_plasmid.png
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[[File:T--NUS Singapore--Construct_tested_with_NLS-VP16-EL222_expressed_from_an_episomal_plasmid.png|700px|thumb|center|Figure 1: Construct tested, with NLS-VP16-EL222 expressed from an episomal plasmid containing mKO under the control of a CYC1 core promoter with a C120 motif upstream. ]]
  
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https://static.igem.org/mediawiki/parts/thumb/a/a0/T--NUS_Singapore--BY4741_wildtype_compared_to_BY4741_containing_pC120-promoter.png/600px-T--NUS_Singapore--BY4741_wildtype_compared_to_BY4741_containing_pC120-promoter.png
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[[File:T--NUS Singapore--Induction of pC120-mKO-EL-U.png|700px|thumb|center|Figure 1: BY4741 wildtype compared to BY4741 containing pC120-promoter controlling mKO as well as constitutive expression of NLS-VP16-EL222.]]
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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 overtime 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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[[File:T--NUS Singapore--T--NUS_Singapore--Expression_of_C120-mKO-EL222-U_overtime.png|700px|thumb|center|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.]]
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'''Characterization of expression NLS-VP16-EL222 from genome integrated cassette'''
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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).
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The 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 a decrease in expression due to a reduction in copy when NLS-VP16-EL222 is expressed from the genome as compared to an episomal plasmid.
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[[File:T--NUS Singapore--Improvement of blue light system.png|700px|thumb|center|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.]]
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'''Use of EL222 as a repressible factor'''
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It has been shown that in E.coli, NLS-VP16-EL222 can function as a blue light-inducible repressor if the C120 motif is placed in proximity to the TATA box of a promoter, as in the presence of blue light it binds to the motif and sterically hinders the formation of the transcription initiation complex[2]. Two similar constructs were tested in BY4741, where either 3 C120 repeats or 1 C120 were placed downstream of the TATA box of a PGK1p, and this modified promoter was placed upstream of a mTurquoise fluorescent gene(Figure 5).
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https://static.igem.org/mediawiki/parts/thumb/8/85/T--NUS_Singapore--Promoter_constructs_for_testing_NLS-VP16-EL222%E2%80%99s_utility_as_a_repression_factor.png/800px-T--NUS_Singapore--Promoter_constructs_for_testing_NLS-VP16-EL222%E2%80%99s_utility_as_a_repression_factor.png
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Testing with construct containing 3 C120 repeats showed minimal expression compared to the unedited PGK1p, when transformed into BY4741 containing no EL222 expression, activity of the promoter was not restored, indicating that 3xC120 permanently disabled the promoter independent of NLS-VP16-EL222 expression(Figure 6)
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https://static.igem.org/mediawiki/parts/thumb/f/f2/T--NUS_Singapore--RFU_measured_for_pRepress-1_without_NLS-VP16-EL222.png/800px-T--NUS_Singapore--RFU_measured_for_pRepress-1_without_NLS-VP16-EL222.png
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When number of repeats was reduced to 1, promoter activity was restored, but showed no light inducible expression(Figure 7). Thus, so far we have tested that with these constructs cannot replicate the same mechanism of NLS-VP16-EL222 mediated blue light repression that have been shown in E.coli.
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https://static.igem.org/mediawiki/parts/thumb/4/4e/T--NUS_Singapore--pRepress-2_construct_expressed_alongside_NLS-VP16-EL222.png/800px-T--NUS_Singapore--pRepress-2_construct_expressed_alongside_NLS-VP16-EL222.png
  
 
===User Reviews===
 
===User Reviews===

Latest revision as of 12:10, 19 October 2021


This experience page is provided so that any user may enter their experience using this part.
Please enter how you used this part and how it worked out.

Applications

Applied use of BBa_K3570021 by the Team NUS Singapore 2021

As this part has not been successfully cloned and tested yet, Team NUS Singapore 2021 has performed basic assembly and characterization of this construct as part of our contribution to the registry. We have also made use of this part for our optogenetic project for iGEM 2021, and more detailed descriptions of how our team has utilized this part can be found at our team wiki page: https://2021.igem.org/Team:NUS_Singapore

Characterization of expression NLS-VP16-EL222 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 a minimal C120 containing promoter 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 1: Construct tested, with NLS-VP16-EL222 expressed from an episomal plasmid containing mKO under the control of a CYC1 core promoter with a C120 motif upstream.
Figure 1: 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 overtime 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.

File:T--NUS Singapore--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 NLS-VP16-EL222 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).

The 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 a decrease in expression due to a reduction in copy when NLS-VP16-EL222 is expressed from the genome as compared to an episomal plasmid.

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.

Use of EL222 as a repressible factor It has been shown that in E.coli, NLS-VP16-EL222 can function as a blue light-inducible repressor if the C120 motif is placed in proximity to the TATA box of a promoter, as in the presence of blue light it binds to the motif and sterically hinders the formation of the transcription initiation complex[2]. Two similar constructs were tested in BY4741, where either 3 C120 repeats or 1 C120 were placed downstream of the TATA box of a PGK1p, and this modified promoter was placed upstream of a mTurquoise fluorescent gene(Figure 5).

800px-T--NUS_Singapore--Promoter_constructs_for_testing_NLS-VP16-EL222%E2%80%99s_utility_as_a_repression_factor.png

Testing with construct containing 3 C120 repeats showed minimal expression compared to the unedited PGK1p, when transformed into BY4741 containing no EL222 expression, activity of the promoter was not restored, indicating that 3xC120 permanently disabled the promoter independent of NLS-VP16-EL222 expression(Figure 6)

800px-T--NUS_Singapore--RFU_measured_for_pRepress-1_without_NLS-VP16-EL222.png

When number of repeats was reduced to 1, promoter activity was restored, but showed no light inducible expression(Figure 7). Thus, so far we have tested that with these constructs cannot replicate the same mechanism of NLS-VP16-EL222 mediated blue light repression that have been shown in E.coli.

800px-T--NUS_Singapore--pRepress-2_construct_expressed_alongside_NLS-VP16-EL222.png

User Reviews

UNIQ7d787997afbb63ac-partinfo-00000000-QINU UNIQ7d787997afbb63ac-partinfo-00000001-QINU