Difference between revisions of "Part:BBa K1033225:Experience"
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This experience page is provided so that any user may enter their experience using this part.<BR>Please enter | This experience page is provided so that any user may enter their experience using this part.<BR>Please enter | ||
how you used this part and how it worked out. | how you used this part and how it worked out. | ||
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| + | ===Utah State 2015 iGEM Team Use=== | ||
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| + | <p>The Utah State 2015 iGEM Team used this, along with two other related promoters (CP8, CP11, CP44) in a ''Lactococcus lactis'' project. Due to time constraints and difficulties working with non-model organisms, the constructs using these promoters were not inserted into ''L. lactis'' but were cloned into ''Escherichia coli'' and compared in strengths using the sfGFP(Bs) and mCherry(Lr) fluorescent proteins (see Figures 1 and 2, respectively). The findings were not consistent across different fluorescent proteins, which may be due to secondary structure differences in mRNA or in the protein sequences themselves. These data were very consistent for each of the six constructs over several biological and technical replicates.</p> | ||
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| + | https://static.igem.org/mediawiki/2015/a/ad/Utah_State_2015_sfGFP%28Bs%29_Fluorescence_Chart_Version3.jpeg | ||
| + | <p>Figure 1. Fluorescence levels from three constructs using synthetic promoters from Jensen and Hammer (1998) in E. coli excited at 485/20 with emissions read at 528/20. cp8 from part BBa_K1820014, cp11 from part BBa_K1820015, and cp44 from part BBa_K1820016.</p> | ||
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| + | https://static.igem.org/mediawiki/2015/0/0b/Utah_State_2015_MCherry_Fluorescence_Chart_Version_2.jpg | ||
| + | <p>Figure 2. Fluorescence levels from three constructs using synthetic promoters from Jensen and Hammer (1998) in E. coli excited at 530/25 with emissions read at 590/35. cp8 from part BBa_K1820017, cp11 from part BBa_K1820018, and cp44 from part BBa_K1820019</p> | ||
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| + | <p> Referrence: Jensen, P. R., Hammer, K. (1998). The Sequence of Spacers between the Consensus Sequences Modulates the Strength of Prokaryotic Promoters. Appl Environ Microbiol. 1998 Jan; 64(1): 82–87. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC124675/ </p> | ||
===Applications of BBa_K1033225=== | ===Applications of BBa_K1033225=== | ||
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| + | <I>BABS UNSW 2015</I> | ||
| + | <br>A LacZ assay was carried out with p170 (<a href="https://parts.igem.org/Part:BBa_K1677300">BBa_K1677300</a>), CP44 (<a href="https://parts.igem.org/Part:BBa_K1033225">BBa_K1033225</a>) and ASR (<a href="https://parts.igem.org/Part:BBa_K1033225">BBa_K1033225</a>) fused to a promoter activity reporter (BBa_I732095) in E. Coli and grown in LB with antibiotics for 8 hours. The data normalized to OD600 of the cultures reveals a decrease in lacZ production correlated with a decrease in pH for the CP44 promoter. A slight increase was observed in both p170 and ASR with the decline in pH, however expression of lacZ is much lower even at neutral pH than with CP44. </br> <br> This characteristic of lowered expression in acidic conditions has previously not been observed with this promoter </br> | ||
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| + | <figure><center><img src="https://static.igem.org/mediawiki/2015/a/af/Unsw2015_laczassay_ph_odnormalised_graph.jpeg" style="PADDING-BOTTOM:0%;PADDING-TOP:0%; max-width:70%;"></center></figure> | ||
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| + | <br> When the above data is normalized to the neutral pH value of the culture the differential expression at varying pH values becomes more pronounced. | ||
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| + | <figure><center><img src="https://static.igem.org/mediawiki/2015/a/ac/Unsw2015_laczassay_ph_column_graph.jpeg" style="PADDING-BOTTOM:0%;PADDING-TOP:0%; max-width:70%;"></center></figure> | ||
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| + | </html> | ||
===User Reviews=== | ===User Reviews=== | ||
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[[File:Graphricksfgfpbs.png]] | [[File:Graphricksfgfpbs.png]] | ||
| − | The graph shown above helps us to know distribution of the fluorescence in the cells of E.coli with different CP promotor fused to superfoldedGFP-terminator-RBS collection (CP1, CP11, CP29, CP30, CP41, CP44) and J23101 as reference. We used this method to characterize the CP promoter collection that was BioBricked by the Uppsala 2013 team. | + | The graph shown above helps us to know distribution of the fluorescence in the cells of E.coli with different CP promotor fused to superfoldedGFP-terminator-RBS collection (CP1, CP11, CP29, CP30, CP41, CP44) and J23101 as reference. We used this method to characterize the CP promoter collection that was BioBricked by the Uppsala 2013 team. |}; |
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Latest revision as of 00:46, 27 September 2015
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.
Utah State 2015 iGEM Team Use
The Utah State 2015 iGEM Team used this, along with two other related promoters (CP8, CP11, CP44) in a Lactococcus lactis project. Due to time constraints and difficulties working with non-model organisms, the constructs using these promoters were not inserted into L. lactis but were cloned into Escherichia coli and compared in strengths using the sfGFP(Bs) and mCherry(Lr) fluorescent proteins (see Figures 1 and 2, respectively). The findings were not consistent across different fluorescent proteins, which may be due to secondary structure differences in mRNA or in the protein sequences themselves. These data were very consistent for each of the six constructs over several biological and technical replicates.
Figure 1. Fluorescence levels from three constructs using synthetic promoters from Jensen and Hammer (1998) in E. coli excited at 485/20 with emissions read at 528/20. cp8 from part BBa_K1820014, cp11 from part BBa_K1820015, and cp44 from part BBa_K1820016.
Figure 2. Fluorescence levels from three constructs using synthetic promoters from Jensen and Hammer (1998) in E. coli excited at 530/25 with emissions read at 590/35. cp8 from part BBa_K1820017, cp11 from part BBa_K1820018, and cp44 from part BBa_K1820019
Referrence: Jensen, P. R., Hammer, K. (1998). The Sequence of Spacers between the Consensus Sequences Modulates the Strength of Prokaryotic Promoters. Appl Environ Microbiol. 1998 Jan; 64(1): 82–87. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC124675/
Applications of BBa_K1033225
BABS UNSW 2015
A LacZ assay was carried out with p170 (BBa_K1677300), CP44 (BBa_K1033225) and ASR (BBa_K1033225) fused to a promoter activity reporter (BBa_I732095) in E. Coli and grown in LB with antibiotics for 8 hours. The data normalized to OD600 of the cultures reveals a decrease in lacZ production correlated with a decrease in pH for the CP44 promoter. A slight increase was observed in both p170 and ASR with the decline in pH, however expression of lacZ is much lower even at neutral pH than with CP44.
This characteristic of lowered expression in acidic conditions has previously not been observed with this promoter
When the above data is normalized to the neutral pH value of the culture the differential expression at varying pH values becomes more pronounced.
User Reviews
UNIQe3b9b74710ecb594-partinfo-00000001-QINU
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No review score entered. Username |
This part was used in the composite part BBa_K1365555 for characterization by the Rijksuniversiteit Groningen.
The graph shown above helps us to know distribution of the fluorescence in the cells of E.coli with different CP promotor fused to superfoldedGFP-terminator-RBS collection (CP1, CP11, CP29, CP30, CP41, CP44) and J23101 as reference. We used this method to characterize the CP promoter collection that was BioBricked by the Uppsala 2013 team. |};
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