Difference between revisions of "Part:BBa K3889100"

 
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<partinfo>BBa_K3889100 short</partinfo>
 
<partinfo>BBa_K3889100 short</partinfo>
  
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Composite part for SRTF expression and fluorescence check-in bacillus
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SRTF1 is expressed under a constitutive promoter SP126 (<partinfo>BBa_K3889010</partinfo>).
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In absence of progesterone it can bind to its binding site (<partinfo>BBa_K3889030</partinfo>) and repress transcription of the gene. Now as progesterone enters the system SRTF1 binds to progesterone leaving its binding site free for the transcription machinery to start working. This results in a progesterone inducible gene expression.
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mcherry was linked with the help of P2A linker (<partinfo>BBa_K3889069</partinfo>)in order to coexpress it with SRTF1 and check protein expression
  
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<!-- Add more about the biology of this part here
 
 
===Usage and Biology===
 
===Usage and Biology===
  
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<!-- -->
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This part along with <partinfo>BBa_K3889101</partinfo> and <partinfo>BBa_K3889102</partinfo> was used in order to produce Ovastacin (<partinfo>BBa_K3889022</partinfo>) when progesterone in absent in the fallopian tube. The working of the system is shown below:
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<html>
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<body>
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<video style="width: 100%" muted id="myVideo" controls autoplay loop>
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<source src="https://static.igem.org/mediawiki/parts/c/c7/T--IISER-Tirupati_India--ovastacin_production.mov">
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</video>
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</html>
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===Results===
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[[File:T--IISER-Tirupati India--Progesterone sensing in SRTF1-P22 Combined Cassette.png|centre|900 px]]
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In this graph, SRTF1 cassette (BBa_K3889100) and P22 Cassette(BBa_K3889101) were co-expressed in <i>Bacillus subtilis</i> 168 consisting of SRTF1 with mCherry reporter and P22 Cassette with Azurite reporter under various concentrations of progesterone.
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Unexpectedly, the mCherry expression decreased at higher concentrations of progesterone. Further experimental evidence is needed to determine the cause. Additionally, the Azurite expression increased with progesterone levels signifying that p22 protein production increased. Hence, there is evidence to suggest that SRTF1 and SRTF1 DNA binding sites were interacting as expected and progesterone sensing is taking place in <i>Bacillus subtilis</i>.
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<strong><u>Results Table</u></strong>:
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{| {{table}}
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!Progesterone Conc. (in nM)
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!Azurite
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!mCherry
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|-
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| 0||11374||297821
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|-
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| 16.5||17101||281770
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|-
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| 33||17275||287161
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|-
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| 82.5||29714||249459
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|-
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| 165||45536||153234
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|-
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| 330||78069||117241
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|}
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<span class='h3bb'>Sequence and Features</span>
 
<span class='h3bb'>Sequence and Features</span>
 
<partinfo>BBa_K3889100 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K3889100 SequenceAndFeatures</partinfo>

Latest revision as of 00:24, 22 October 2021


SRTF1 expression cassette Bacillus subtilis

SRTF1 is expressed under a constitutive promoter SP126 (BBa_K3889010). In absence of progesterone it can bind to its binding site (BBa_K3889030) and repress transcription of the gene. Now as progesterone enters the system SRTF1 binds to progesterone leaving its binding site free for the transcription machinery to start working. This results in a progesterone inducible gene expression.

mcherry was linked with the help of P2A linker (BBa_K3889069)in order to coexpress it with SRTF1 and check protein expression

Usage and Biology

This part along with BBa_K3889101 and BBa_K3889102 was used in order to produce Ovastacin (BBa_K3889022) when progesterone in absent in the fallopian tube. The working of the system is shown below:

Results

T--IISER-Tirupati India--Progesterone sensing in SRTF1-P22 Combined Cassette.png

In this graph, SRTF1 cassette (BBa_K3889100) and P22 Cassette(BBa_K3889101) were co-expressed in Bacillus subtilis 168 consisting of SRTF1 with mCherry reporter and P22 Cassette with Azurite reporter under various concentrations of progesterone.

Unexpectedly, the mCherry expression decreased at higher concentrations of progesterone. Further experimental evidence is needed to determine the cause. Additionally, the Azurite expression increased with progesterone levels signifying that p22 protein production increased. Hence, there is evidence to suggest that SRTF1 and SRTF1 DNA binding sites were interacting as expected and progesterone sensing is taking place in Bacillus subtilis.

Results Table:

Progesterone Conc. (in nM) Azurite mCherry
0 11374 297821
16.5 17101 281770
33 17275 287161
82.5 29714 249459
165 45536 153234
330 78069 117241

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]