Part:BBa_M33330
RC of atzR CDS
Pseudomonas sp. ADP digests atrazine under nitrogen starvation conditions. atzABC are constitutively expressed and convert atrazine to cyanuric acid. atzDEF convert cyanuric acid to carbon dioxide and ammonia, which the cell digests. The atzDEF operon is regulated by atzR, a protein that autoregulates its production in the same region. GlnK is produced under nitrogen limitation conditions and induces expression of atzDEF.
This part should be used in conjunction with BBa_M33333, the atzDEF operator region. See related parts BBa_M33331, BBa_M33332, and BBa_M33333.
Improved by Shanghai_city, 2020
According to the registry, the part BBa_M33330 designed by group Stanford BIOE44 - S010, described a regulated circuit about atzR. However, there was no any experiment results or wiki documentation in support of its function. Thus, we consider it is meaningful to conduct a series of experiments to explore the function of atzR.
Our team iGEM20_Shanghai_city designed a new composite part BBa_K3526006. Compared with the existing part BBa_M33330, we optimized the coding sequence of atzR and added corresponding parts, such as a constitutive-promoter tac, a regulated-promoter PatzD, a reporter EGFP, and a terminator T7 (Fig4). Then, our new composite part BBa_K3526006 was given a completely new function, detection of the presence of cyanuric acid by observed green fluorescence. Our experiment data also supported the functionality of our system.
Contribution
BBa_K3526006 is a composite part contains a special transcription factor AtzR and a special promoter PatzD. The transcription factor AtzR can sense the presence of cyanuric acid (CYA), and then regulate the promoter PatzD to start expressing GFP protein. As a result, at the presence of cyanuric acid, the engineering strain with BBa_K3526006 can emit green fluorescence.
Engineering Success
The cyanuric acid (CYA) was used for functional testing. After reading related articles, we choose 0μM, and 50μ as the detection concentrations of CYA. After preliminary analysis, we obtained Figure 3.
As seen in Figure 3, when the Pseudomonas putida is not transformed, under different concentrations of CYA, the GFP fluorescence value does not change significantly along with time. However, when part BBa_K3526006 is transformed, the GFP fluorescence value increases along with time when the concentration of CYA is 50μM. This can be explained as follow: as time goes on, more and more AtzR is transcribed. PatzD is activated when atzR combines CYA leading to more transcription of GFP. The results of the experiment show that with the presence of part BBa_K3526006,Pseudomonas putida KT2440 can detect cyanuric acid.
The results of the experiment have proven that by expression of BBa_K3526006, Pseudomonas putida KT2440 can effectively detect the presence of cyanuric acid (CYA) from concentrations 0μM to 50μM. It is reported that atrazine can be degraded into cyanuric acid via AtzA/AtzB/AtzC enzyme, and the detection of atrazine degradation can also be done. We imagine and design to conduct a composite part contains the coding sequence of the AtzA/AtzB/AtzC enzyme. Based on our results of BBa_K3526006, we can co-transform the new composite part and BBa_K3526006 into Pseudomonas putida KT2440. The new improved engineering strain can degrade atrazine and produce cyanuric acid, then the transcription factor AtzR can sense the cyanuric acid and regulate the PatzD promoter to express the GFP protein. As a result, we complete using the improved composite part to detect the presence of atrazine.
Edited by Shanghai_city, 2020
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 34
Illegal AgeI site found at 196 - 1000COMPATIBLE WITH RFC[1000]
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