Difference between revisions of "Part:BBa K1350001"

Latest revision as of 14:35, 15 October 2014

A kind of secretory protein named kil

This is a part.Its sequence length is 136bp and it begins with ATG.It will express the protein, and role in the periplasmic space of e. coli. n wild-type E.coli exist a plasmid named colE1, the plasmid encodes colicin gene, the release of colicin gene, immune gene of Escherichia coli in chain( in plasmid colE1 were known as CEA, kil, Imm gene). The expression of kil gene can promote the colicin release to kill the other strain . This is because the expression of kil gene can significantly increase the bacterial outer membrane permeability, so as to effectively promote the secretion of periplasmic colicin secreted to the extracellular.

Verification of kil secretion

We connected the kil gene on the downstream of alkaline cellulase , and through the induction of IPTG, successfully proved that kil has the effect of secreting exogenous protein.
To measure the ability of kil to exogenous protein secretion of alkaline cellulase，we determine the activity of alkaline cellulase by DNS colorimetric method.
As shown in the figure, the more deeper color represents the more amount of alkaline cellulase secreting out of the Escherichia coli, which can express the secretion ability of kil is strong.

Figure 1.Determination of enzyme activity in cell supernatant and cells
Lane 1.enzyme activity of cell supernatant with kil
Lane 2.enzyme activity of cells with kil
Lane 3.enzyme activity of cell supernatant without kil
Lane 4.enzyme activity of cells without kil

Figure 5.Determination of enzyme activity in cell supernatant
Lane 1.enzyme activity of cell supernatant without kil

Lane 3.enzyme activity of cell supernatant with kil

The above figures show that kil can secrete the Exogenous protein,but the mechanism of Kil protein increasing the permeability of bacterial outer membrane is not very clear. There is evidence that phospholipase A involves several links of kil secretion.The periplasmic protein secreting out of cell has many benefits. First of all, extracellular volume is much larger than the periplasmic space, can accumulate more objective protein. Secondly, the process of purifying extracellular periplasmic proteins is much simpler than the purification of cell wall breaking.

Sequence and Features