Difference between revisions of "Part:BBa K1709002"

Latest revision as of 16:13, 26 September 2015

CheZ-GFP

Upon recognition of a small molecule (e.g. nutrients or toxins), chemoreceptors transduce a signal through a set of Che proteins. This group of methylesterases and phosphatases regulates the rotation of the flagella. At the end of the chain of Che proteins is CheY. In phosphorylated state, CheY binds the flagella and causes the cells to tumble. The phosphatase CheZ dephosphorylates CheY, inducing dissociation of CheY from the flagella enabling the cells to swim. Cells lacking the CheZ protein are unable to swim and will tumble excessively and incessantly.

The sequence was verified by restriction mapping and PCR and the presence of colonies expressing GFP proves that the plasmid was designed and cloned correctly.

Further DNA sequencing proved the correct sequence, as designed.

To characterize the CheZ-GFP BioBrick, the fragment containing a RBS was cloned directly after a strong promotor (BBa_J23101). The first figure shows the gel right before ligation.
The colonies were checked by restriction mapping using BcuI and PstI (results not shown). The DNA sequence was also confirmed by DNA sequencing. Results can be provided by email. The presence of colonies expressing GFP proves that the plasmid was designed and cloned correctly.

Further DNA sequencing proved the correct sequence, as designed.

Part of the sequence alignment with on top the reference sequence and on the bottom the sequencing results.

Gel after purification. Lanes 2-5: insert (1400bp). Lane 6: linearized vector. Lanes 7-10 : insert. Lane 11: linearized vector

GFP is expressed in the cells. This confirms the correct construction of the BioBrick

Further characterization could be done by transforming the cheZ knockout Keio strain with this plasmid. These cells should then regain their possibility to swim.

Sequence and Features