Part:BBa_K3799057
Bidirectional AIP-1 sensor
This is an improvement of the existing part BBa_K1022100.
Usage and Biology
This is a high throughput sensing module that can sense the presence of AIP-I molecules. This part consists of a pBAD promoter, an AIP sensor infrastructure (BBa_I746101), an AIP inducible promoter P2 and a GFP reporter.
In the natural system, the signalling Auto-inducing peptide (termed AIP) is made from AgrD, while AgrB, a transmembrane protein that is responsible for the cyclization. AgrC and AgrA, form the two-component system responsible for AIP detection and response relay, respectively. Upon binding to AIP, AgrC, a transmembrane histidine kinase receptor, facilitates the phosphorylation of the transcriptional activator AgrA. The phosphorylated AgrA, in turn, activates gene expression regulated by P2 and P3 promoters. There are four known variants of AIP (AIP I-IV) with different molecular structures and cross-inhibitory activity.
This part has been improved from BBa_K1022100 by flipping the direction of p2 promoter to the opposite direction of pBAD promoter.
In the existing part, we observed a significant increase in GFP expression in samples with zero AIP concentration. Based on this observation, we inferred that this could be due to the leaky gene expression of GFP. We have improved the existing part in such a way that the leaky gene expression reduced significantly.
Design
To solve the leaky gene expression we designed a bidirectional circuit. We Used Hi-Fi DNA assembly kit to make this bidirectional circuit. First, we divided the existing part (BBa_K1022100) into three segments and ordered those from Twist Bioscience. These three fragments were assembled in two different ways to produce the existing part as well as our designed improved part. For these assemblies, we designed two different sets of primers (a total of eight primers) using the NEBilder tool. We extended our three gene fragments with appropriate primers to produce overlap sequences at both ends of each fragment.
To make the improved part, we designed the overlap sequences in such a way that the p2 promoter along with the GFP reporter is flipped. This will make the direction of pBAD promoter and P2 promoter opposite to each other, which will significantly decrease the leaky gene expression of GFP reporter.
Charectarization
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 1105
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 1493
Illegal BamHI site found at 1045
Illegal BamHI site found at 2377 - 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 295
Illegal AgeI site found at 720 - 1000COMPATIBLE WITH RFC[1000]
Functional Parameters
| None |