Part:BBa_K2912015
Trp_Lysis gene
Trp_Lysis gene expression can be controlled by the concentration of Tryptophan
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 7
Illegal NheI site found at 30 - 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
Usage and Biology
The lysis gene from colicin-producing strains of bacteria encodes the lysis protein which is only 4872Da in molecular weight and performs two functions that are instrumental to the activity of Colicin E7 (ColE7):one of its function is to Release of ColE7 from ColE7—Immunity complex and the other is Partial lysis of host cell membrane. On the basic with its special biological function, it's likely to use lysis protein as a cellular structure breaker. In order. In this year, our team decided to add a Tryptophan attenuator between a RBS and the lysis gene, making it possible to switch off or switch on the lyse metabolism by controlling the tryptophan concentration, so that the lysis protein can better serve in our project when we need to break our engineering cells to extract our product.
Release of ColE7 from ColE7—Immunity complex can cause the host cell to lyse as long as the protein is activated. On the basic of its biological function, we can use lysis gene to break our engineering E.coli and release the interior cell contents when it's time to extract our product-shRNA by adding a Tryptophan attenuator between a RBS and lysis gene.
Before the official experiment of Trp_Lysis gene. We have done the T7 promotor_lysis gene characterization experiment with IPTG inducing in LB liquid medium(illustrated with Fig.2).
According to the characterization experiment's result, we can draw a conclusion that once the lysis protein is produced by IPTG inducing, their growth will be significantly inhibited.
In our next step, we transformed the expression vector into HT115 (DE3) E.coli by heat-shock method, culturing in LB liquid medium to exact exponential phase. Afterwards, we started a cell growth curve experiment of our engineering E.coli in different tryptophan concentration gradients and then we calculated their survival rate by use (illustrated with Fig.3).
Besides, we also did a culturing in LB solid medium experiment in different tryptophan concentration gradients.(illustrated with Fig.4)
In contrast with T7 promotor_lysis with IPTG inducing group(illustrated with Fig.5).
Compared to T7 promotor-lysis, the Trp_Lysis gene obviously has the advantage which is able to kill the host cell by breaking their structure in a controlled way. In other words, we can switch off or switch on the killing metabolism by controlling the tryptophan concentration of the culture medium, and the concentration critical valve is around 0.3%(if the concentration is more than 0.3%, the killing metabolism will be closed so that the host cells can grow as usual)
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