Difference between revisions of "Part:BBa K3510005"

 
 
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<partinfo>BBa_K3510005 short</partinfo>
 
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<b><font color= red>Important: This construct does not include a RBS upstream of the FAST2 sequence. When building on our design please make sure you add it in order to allow for translation</font color= red></b> 
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This composite part is a manganese inducible expression system of a phytochelatin (BBa_K1321005) tagged with FAST2 (BBa_K3510000). Expression is controlled by two individual elements: A strong Anderson promoter (BBa_J23102) and a manganese riboswitch (BBa_K902074). With this promoter choice transcription will constitutively happen, but translation initiation is dependent on manganese binding to the riboswitch. Transcriptional termination occurs through the activity of the reliable double terminator (BBa_B0015). This construct allows to assess the efficiency of the riboswitch by itself, without the support of the manganese promoter (contained inBBa_K3510003). 
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<b>Usage:</b> In our project we coupled this manganese sensing device with a phytochelatin in order to create a bifunctional biosensor, that is not only able to detect manganese, but also directly binds it. With our suggested implementation this would contribute to the increase of water quality. The FAST2 tag allows a quantitative measurement of the fluorescence, which should provide information about the manganese concentration in the sample.
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The cloning of this composite part was successful and the steps were perfected (see our notebook) to increase the efficiency (<b>Fig. 1</b>). Due to the missing RBS in our design, the results from our experiments dependent on protein expression (fluorescence, growth, etc.) are omitted here (see our experiments and results page if interested in experimental design), as they do not represent the potential functionality of our system.
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<html><p><img src="https://2020.igem.org/wiki/images/b/b4/T--Tuebingen--ColonyPCR_GA2-GA4-GA3.png" alt="Colony PCR" width="600px"></p></html>
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<b>Figure 1: Colony PCR after Gibson Assembly of Anderson-Promoter-Mn-Riboswitch-FAST-Phytochelatine-Terminator (GA4).</b> This figure is an example for the high cloning efficiency when using Gibson Assembly. It also contains samples from two other composite parts (GA2 and GA3).
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===Sequence and Features===
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<partinfo>BBa_K3510005 SequenceAndFeatures</partinfo>
  
 
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<span class='h3bb'>Sequence and Features</span>
 
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Latest revision as of 20:36, 26 October 2020

Anderson-Promoter-Mn-Riboswitch-FAST-Phytochelatine-Terminator

Important: This construct does not include a RBS upstream of the FAST2 sequence. When building on our design please make sure you add it in order to allow for translation

This composite part is a manganese inducible expression system of a phytochelatin (BBa_K1321005) tagged with FAST2 (BBa_K3510000). Expression is controlled by two individual elements: A strong Anderson promoter (BBa_J23102) and a manganese riboswitch (BBa_K902074). With this promoter choice transcription will constitutively happen, but translation initiation is dependent on manganese binding to the riboswitch. Transcriptional termination occurs through the activity of the reliable double terminator (BBa_B0015). This construct allows to assess the efficiency of the riboswitch by itself, without the support of the manganese promoter (contained inBBa_K3510003).

Usage: In our project we coupled this manganese sensing device with a phytochelatin in order to create a bifunctional biosensor, that is not only able to detect manganese, but also directly binds it. With our suggested implementation this would contribute to the increase of water quality. The FAST2 tag allows a quantitative measurement of the fluorescence, which should provide information about the manganese concentration in the sample.

The cloning of this composite part was successful and the steps were perfected (see our notebook) to increase the efficiency (Fig. 1). Due to the missing RBS in our design, the results from our experiments dependent on protein expression (fluorescence, growth, etc.) are omitted here (see our experiments and results page if interested in experimental design), as they do not represent the potential functionality of our system.

Colony PCR

Figure 1: Colony PCR after Gibson Assembly of Anderson-Promoter-Mn-Riboswitch-FAST-Phytochelatine-Terminator (GA4). This figure is an example for the high cloning efficiency when using Gibson Assembly. It also contains samples from two other composite parts (GA2 and GA3).


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 7
    Illegal NheI site found at 30
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 147