Team:SCUT China/Basic Part
1.Usage and Biology:
GadB is an acid tolerant factor which play an important role in the acid tolerance of E.coli MG1655.The decarboxylation of glutamate consumes a proton, and therefore, micro‐organisms take advantage of this property to remove protons from the intracellular milieu under acidic conditions[1]. GadB is involved in the AR system, which is the glutamic acid-dependent acid resistance (GDAR) system, consisting of the homologous inducible glutamic acid decarboxylases GadA/GadB enzymes and the glutamate/γ-aminobutyric acid (GABA) antiporter GadC[2].
2.Characterization:
We have expressed this gene and tested its 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) to test our VerProS system.
The functional gene gadB 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(+)-gadB 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.1) which shows that target protein has been expressed successfully.
Fig.1 The SDS-PAGE of gadB and ybaS
What’s more, we have tested the Protein expression of gadB. 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.2 Multiple regression curve of protein concentration
Finally, the expression of gadB was calculated as 0.0342 mg/ml.
The last, we have tested its influence on the acid tolerance of MGR. MG, MGR and MGR expressing gadB 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 gadB or not on acid tolerance. Under moderate acid stress, the final OD600 value of strain MGR-gadB(the strain overexpressing gadB) was 44.2% higher than that of the wild type strain (MG) (Fig. 3).
Fig.3 Growth of strains MG,MGR and MGR-gadB under acid stress.
Other Basic Parts
T7 Promoter:
PT7 is the most well-known inducible promoter with high transcriptional strength. T7 RNAP is a single-subunit RNA polymerase that is a strong driver of transcription. It is functionally orthogonal to most hosts, acting only on its cognate promoter, PT7.
From the research of Ghodasara A et al[3], we found different strength of PT7 and made full use of them for our VerProS pool. How our VerProS pool work you can learn from it.
In order to achieve a larger scale and more accurate regulation range, we have improved T7 promoters with different strength providing more options for the precise regulation.
Here are our T7 promoter variants family member.
Fig 4: Information of T7 promoter variants family member
Toehold Switch & Trigger DNA:
Toehold switch is prokaryotic riboregulators that activate gene expression in response to cognate RNAs( Trigger RNAs) with arbitrary sequences [3], we have added Toehold switch A&C and their Trigger DNAs (Toehold switch B&D is from previous part number range).
Acid Tolerant Factor:
YbaS is a glutaminase that mediates an acid tolerance system. This acid tolerance system in E. coli that relies on L-glutamine (Gln), one of the most abundant food-borne free amino acids. Upon uptake into E. coli, Gln is converted to L-glutamate (Glu) by the acid-activated glutaminase YbaS, with concomitant release of gaseous ammonia [4]. The free ammonia neutralizes proton, resulting in elevated intracellular pH under acidic environment. Not only that, YbaS and the amino acid antiporter GadC, which exchanges extracellular Gln with intracellular Glu, together constitute an acid resistance system that is sufficient for E. coli survival under extremely acidic environment.
An amino acid antiporter GadC exchanges extracellular L-glutamate (Glu) with intracellular γ-aminobutyric acid (GABA), but also Gln and L-methionine. GadC can work synergistically with a variety of acid-resistant factors to form an acid-resistant system. It is known that YbaS-GadC is a glutamine-dependent acid-resistant system and GDAR(AR2) is glutamic acid-dependent acid-resistant system [2].
KatA is catalase. Microorganisms induce the production of harmful reactive oxygen species (ROS) under acid stress. Overexpression of katA results in a significant decrease in intracellular ROS levels [5]. KatA-mediated ROS clearance plays an important role in conferring resistance to low pH stress in C. glutamicum cells.
Reference
[1] Feehily C, Karatzas KAG. Role of glutamate metabolism in bacterial responses towards acid and other stresses. J Appl Microbiol. 2013;114:11–24. doi: 10.1111/j.1365-2672.2012.05434.x.
[2] Kanjee U, Houry WA. Mechanisms of acid resistance in Escherichia coli. Annu Rev Microbiol. 2013;67:65–81. 10.1146/annurev-micro-092412-155708
[3] Green AA, Silver PA, Collins JJ, Yin P. Toehold switches: de-novo-designed regulators of gene expression. Cell. 2014;159(4):925–939. doi:10.1016/j.cell.2014.10.002
[4]Lu P, Ma D, Chen Y, et al. L-glutamine provides acid resistance for Escherichia coli through enzymatic release of ammonia. Cell Res. 2013;23(5):635-44.
[5] Xu, N., Lv, H., Wei, L. et al. Impaired oxidative stress and sulfur assimilation contribute to acid tolerance of Corynebacterium glutamicum. Appl Microbiol Biotechnol (2019).