Difference between revisions of "Part:BBa K1675002"

Revision as of 14:36, 20 October 2019

glsA, glutaminase1

GlsA encodes an acid-activated glutaminase, which is sufficient for an acid resistance system, and it is able to catalyze a reaction converting glutamate to glutamine and releasing the free gaseous ammonia. The free gaseous ammonia will consume the proton and increase the intracellular pH (Fig.1). The robust glutaminase activity only exists at pH 6.0 or lower. The highest activity is obtained at pH 4.0, followed by pH 5.0 and 6.0. In contrast, GlsA is not activated at pH 7.0 and 8.0. The dependence on pH makes it an effective tool to respond when necessary.

Fig.1 The mechanism of GlsA

The difference between the testing group and the control group is not evident. We employed a strong promoter T7 to check whether the protein has been expressed. The functional gene GlsA was constructed on plasmid pET28a and the plasmid was transformed into BL21(DE3). 0.5% IPTG was added to induce the expression of the protein. The following is the picture of SDS-PAGE (Fig.2). It shows that our target protein has been expressed successfully.

Fig.2 The SDS-PAGE of GlsA and pET28a

Characterization from SCUT_China 2019:

GlsA is an acid tolerant factor also named ybaS which has a great influence on the acid resistance of the strain. SCUT_China 2019 have expressed this gene and tested their influence on the acid tolerance of E.coli MG1655-T7 RNAP (MGR). T7 RNA polymerase was integrated into the genome of E. coli MG1655(MG) by SCUT_China 2019 to test their VerProS system.

The functional gene ybaS was constructed on plasmid pET30a(+) and the plasmid was transformed into MGR. IPTG(0.2 mM) was added to induce the expression of the protein. Inoculated the MGR with pET30a(+)-ybaS in 10ML LB medium,37 ℃，250rpm for 12 hours，and then 1:100 transferred it to 12.5ml medium with IPTG (0.2 mM) for 18 hours. The following is the picture of SDS-PAGE (Fig.3) which shows that target protein has been expressed successfully.

Fig.3 The SDS-PAGE of gadB and ybaS

What’s more, SCUT_China 2019 have tested the Protein expression of ybaS. Using ImageJ for gray scale comparison, the multiple regression curve was drawn with the BSA of 0.0125m to 0.1m as the reference, as follow figure:

Fig.4 Multiple regression curve of protein concentration

Finally, the expression of ybaS was calculated as 0.0665 mg/ml.

The last, SCUT_China 2019 have tested its influence on the acid tolerance of MGR. MG, MGR and MGR expressing ybaS were grown overnight (about 16 h) in LBG medium of pH 7.0 at 37 °C. The cultures were then diluted to initial OD600 0.05 in 300 μL LBG medium of pH 7.0, LBG medium acidified by HCl or succinic acid to pH 4.5. Then the cultures were incubated at 37 °C in 100-well Honeycomb microplates using an automated turbidimeter (Bioscreen C, Oy Growth Curves Ab Ltd., Helsinki, Finland) for online monitoring of OD600 for 24 h. A growth assay under moderate acid stress were performed to investigate the effect of overexpression ybaS or not on acid tolerance. Under moderate acid stress, the final OD600 value of strain MGR-ybaS(the strain overexpressing ybaS) was 53% higher than that of the wild type strain (MG) (Fig. 5).

Fig.5 Growth of strains MG,MGR and MGR-ybaS under acid stress.

Sequence and Features