Part:BBa_K3510002

Mn-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 control consists of two individual elements: the manganese inducible promoter (BBa_K902073) and a manganese riboswitch (BBa_K902074). Transcription is regulated by the promoter, while the riboswitch supports translation initiation only in the presence of manganese. We combined both elements hoping for an even tighter regulation. Transcriptional termination occurs through the activity of the reliable double terminator (BBa_B0015).

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_K3510003. 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 Mn-Promoter-Mn-Riboswitch-FAST-Chromoprotein-Terminator (GA1). This figure is an example for the high cloning efficiency when using Gibson Assembly. It also contains samples from one other composite part (GA3).

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
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 319