Part:BBa_K3510004

Anderson-Promoter-Mn-Riboswitch-FAST-Chromoprotein-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 blue chromoprotein (BBa_K864401) additionally 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 determine the efficiency of the riboswitch by itself, without the support of the manganese promoter (included in construct BBa_K3510002).

Usage: In our project we coupled this manganese sensing device with a chromoprotein to have an easy-to-use biosensor that gives a qualitative signal by color. This allows in field testing without the necessity of a laboratory providing a fluorescence plate reader. Therefore, this construct could be really beneficial to roughly examine water quality in areas with poor infrastructure. Additionally, this construct was designed as a control for the effect of phytochelatin on the sensing mechanism in the construct BBa_K351005. Unlike the phytochelatin the chromoprotein should not interfere with manganese.

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.

Figure 1: Colony PCR after Gibson Assembly of Anderson-Promoter-Mn-Riboswitch-FAST-Chromoprotein-Terminator (GA3). This figure is an example for the high cloning efficiency when using Gibson Assembly. It also contains samples from one other composite part (GA1).

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