Difference between revisions of "Part:BBa K777001"

Revision as of 19:25, 26 September 2012

Tar receptor under the control of constitutive promoter J23100

Fig. 1: Overview of BioBricks containing the Tar gene downstream of Anderson promoters. (Activity according to Berkeley 2006)

The transmembrane chemoreceptor Tar of E. coli mediates chemotaxis towards aspartate and exists as a functional homodimer.

Here, we used a set of 8 constitutive Anderson promoters from the 2006 Berkeley group to test how different levels of constitutive Tar expression affect the chemotaxis of E. coli.

The transcriptional activity of the constructs K777001-K777008 was quantified via quantitative real-time PCR. The results of this experiment can be found in the experience section of the according Anderson promoters. Our data were compared to the activity of the promoters that can be found on the main page of Anderson promoters.
The respective raw dataset can be found in our Team-wiki [http://2012.igem.org/Team:Goettingen/Project/Computational_Data#Raw_Datasets here].

Usage and Biology

This part can be used for chemotaxis assays.

• Here is the complete sequence of this part as an ApE file.

Fig. 2: PYMOL images of Tar with aspartate in its binding pocket (PDB ID: 1WAT). The mutated amino acid residues involved in ligand binding are labeled.

Here, we mutated this part at in total 5 amino acid sites which are important for ligand binding. The mutated sites are the nucleotides for the amino acids at position 69, 73, 149, 150 and 154.

Fig. 3: Sequencing chromatogram excerpt of the generated mutated chemoreceptor Tar_QC library in comparison to the original sequence Tar_QC. On the left, the sequencing chromatogram excerpt represents the original sequence, the right side shows the mutated sequence chromatogram. Sequencing results (top) with mutations for the amino acidsat position 149, 150 and 154 and (bottom) with mutations at 69 and 73. Inserted mutations are indicated by the triplets consisting of N and K, whereby K stands for guanine or thymine, N for any possible base. Only in the mutated regions no distinct peaks are present.G: guanine, C: cytosine, A: adenine, T: thymine.

The sequencing chromatogram of the generated chemoreceptor Tar_QC library revealed a functional mutagenesis. Randomly picked clones indicated a diversity of 2x 10⁵ clones.
















The constructed library was tested in a library selection experiment in which seven new chemical attractants were tested for chemotaxis of the novel generated receptors. As similar chemoreceptors were found to recognize the same respective chemicals, this is evidence for a functional part.



Sequence and Features

Fig. 4: Plasmid map of K777001 in pSB1C3