Difference between revisions of "Part:BBa K3139010"

Revision as of 17:53, 21 October 2019

hasA hemophore coding sequence

hasA is the secreted hemophore of the Serratia marcescens has system. It is secreted by an ABC pathway under iron starvation conditions and is able to acquire heme from various hemoproteins and to deliver it to a specific outer membrane receptor, hasR.

Usage and Biology

The type I secretion pathway (or ATP-binding cassette [ABC] pathway) is one of several pathways used by Gram-negative bacteria to secrete proteins into the extracellular medium. The protein secreted through this system lacks N-terminal signal peptide, and its secretory signal is located in the C-terminal sequence of the protein. The system can secrete proteins from cytoplasm to extracellular without periplasmic intermediate, which are hard to be hydrolyzed by protease.

HasA is the secreted hemophore of the heme acquisition system (Has) of Serratia marcescens Has system. It is secreted by a specific ABC transporter apparatus composed of three proteins: HasD, an inner membrane ABC protein; HasE, another inner membrane protein; and HasF, a TolC homolog. HasA is secreted by an ABC pathway under iron starvation conditions and is able to acquire heme from various hemoproteins and to deliver it to a specific outer membrane receptor, HasR. HasA combined with HasD, which can recognize the C-terminal signal of HasA. It hydrolyzes ATP and open HasF protein channel after receiving the signal. Finally, the system will disintegrate by itself when target protein secret successfully.
Fig.1. The mechanism of HasA system

HasA can be used in Serratia marcescens to secrete certain proteins that we are interested in. For most cases, the only thing we need to do is to link this HasA part to the C-terminal of a target protein, so that the fusion protein can be secreted to the supernatant. In our project, We connected the HasA coding sequence at the C-terminal of fusion protein and TEVpS, so that TEVp is able to cleave the fusion protein outside the cell wall.

Characterize

In order to verify the successful operation and secretion of HasA system in Serratia marcescens 11315, we constructed the RFP-HasA plasmid. Additionally, we constructed the plasmid RFP(without HasA) as a negative control. We successfully transformed the plasmids into S. marcescens by electroporation at an electric voltage of 2100V.

However, we found that we couldn’t screen out the positive transformations with ampicillin, which indicated that our S. marcescens strain has ampicillin resistance. So we changed the plasmid backbone with chloramphenicol resistance to screen out the positive ones. We extracted the plasmids from the pure cultures and sequenced them, which suggested positive results.

Fig.2. Sequencing result of the positive strain.

In order to verify the effect of HasA secretion changing with time, we cultured the positive colonies overnight, and inoculated them in a new liquid LB medium(+chloramphenicol), and tested them for 72 hours with a gradient of four hours. We measured the RFP fluorescence in the supernatant and successfully verified the secretion of HasA, which confirmed that our part does work in S.marcescens.
Fig.3. (A)Measurement of OD600nm at the 60th hour, (B)Measurement of RFP fluorescence intensity at the 60th hour.

Sequence and Features