Difference between revisions of "Part:BBa K3510003"

Revision as of 20:16, 26 October 2020

Mn-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 phytochelatin (BBa_K1321005) 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 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 results page if interested), as they do not represent the potential functionality of our system.

Colony PCR

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

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 327

@@ Line 8: / Line 8: @@
 <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.
+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 results page if interested), as they do not represent the potential functionality of our system.
 <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>
-'''Figure 1: Colony PCR after Gibson Assembly of Mn-Promoter-Mn-Riboswitch-FAST -Phytochelatine-Terminator (GA2). This figure is an example for the high cloning efficiency when using Gibson Assembly. It also contains samples from two other composite parts (GA3 and GA4).
+<b>Figure 1: Colony PCR after Gibson Assembly of Mn-Promoter-Mn-Riboswitch-FAST -Phytochelatine-Terminator (GA2).</b> This figure is an example for the high cloning efficiency when using Gibson Assembly. It also contains samples from two other composite parts (GA3 and GA4).