Difference between revisions of "Part:BBa K1639002"

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GadE

This part is activator of proteins responsible for acid resistance in E.coli. GadE is transcription factor of Gad operon and Gad operon express Glutamate Dependent Acid Resistance genes gadA, gadB, gadC.

Usage and Biology

Escherchia coli Natural Acid Resistance System E. coli possesses four phenotypically distinct systems of acid resistance. These systems: 1. Glutamate dependent acid resistance system (GDAR) 2. Arginine dependent acid resistance system (ADAR) 3. Lysine dependent acid resistance system (LDAR) 4. Ornithine dependent acid resistance system (ODAR)

The most effective one of these systems is the glutamate dependent system so we decided to focus on glutamate dependent system (GDAR).

The GDAR system has in the acid resistance complex pathways. We decided to do the certain pathways that how to do acid resistance with our results of research.

Figure 1: Effects of EvgA, YdeO and GadE overexpression on AR mechanisms

EvgA is the most effective positive regulator of GDAR. (Efficiency: evgA>ydeO>gadE) But evgA takes part in countless number different genes regulation and cellular processes (2,6,7) and also most of this cellular processes are unclear. We cannot predict the results of the overexpression of evgA so we chose gadE and using by overexpression of gadE we can induce glutamate depended acid resistance system.
Briefly talk about the impact of the GadE on the mechanism.

Figure 2

The central activator is the LuxR-family member GadE (formerly known as YhiE). GadE binds to a 20-bp sequence called the gad box, which is located 63-bp upstream of the transcriptional start sites of gadA and gadBC. (4)GadE and GadBox are an important point for GadA and GadBC. This means that GadE and GadBox are an important for acid resistance. The basis section of acid resistance is GadE’s regulation. At least 10 different regulatory take part in this regulation.

GadE have three main activation mechanisms. The first of these is performed by evgA and ydeO. About GadE activation phase:
1.EvgS (sensor kinase) activate EvgA (response regulator)
2.YdeO and evgA are independently from each other and GadE takes an active role in transcription activation.

The second GadE activation circuit includes CRP,RpoS and two AraC-like regulators, GadXand GadW.
These steps are as follows:
1.GadX and GadW, are located downstream of gadA also GadX and GadW directly activate transcription of gadE
2.GadX and GadW also bind to the gadA and gadBC gad box sequences and seem to repress the gadA and gadBC promoters. (4) GadW inhibits this GadX’s repression Also GadX and GadW regulate indirectly GadA/BC transcriptional functions.
3.The balance of power in this circuit is influenced by cAMP and CRP,which together inhibit the synthesis of RpoS. Growth under acidic conditions reduces the concentration of cAMP in the cell. RpoS increases GadX’s transcriptional function The increase in GadX then stimulates transcription of GadE and also down regulates GadW.

The third activation of GadE contains TrmE and glucose. These steps are as follows:
1.The function of TrmE in the cell is not fully defined but it does have a clear effect on tRNA modification
2.TrmE and glucose increase independently GadE’s transcriptional function

We discourse GadE’s regulation and then now we will discourse GadE’s effect mechanisms. GadA/BC’s effect mechanisms to acid resistance are as follows:
1.The external pH is normally neutral but if external pH turns to acidic pH, internal pH begins to become acidic pH with HCl diffusion.
2.If the external pH=2,5 and internal pH begins pH=4.2 ± 0.1. GDAR system will activate for acid resistance.
3.GadC is a transmembrane protein. The external pH begins to change from neutral pH to acidic pH, C-plug (is GadC’s subunit) will open. Then glutamate will take inside.

Figure 3

4.Glutamate in cells convert to GABA by GadB/C that’s glutamate decarboxylase isozymes These steps are as follows:

• This step contains pyridoxal phosphate-containing enzymes that replace the α-carboxyl groups of their amino acid substrates with a proton that is recruited from the cytoplasm.
• HCl that diffusion from outside dissociates H+ and Cl-. This H+ is used and CO² is released by the agency of GadA/B isozymes than this is the last stage of glutamate changes to GABA. Besides this is the most important and the last step for acid resistance.

Figure 4

5.If H+ leaves HCl it is staying back Cl and it will be export from the transport of chlorine channel.
6.The emitted CO² is taken out by diffusion.
7.Finally, the uncovered GABA is thrown out from the transmembrane protein GadC.

Characterization

WESTERN BLOTTING

After cloning GadE into pET45-b successfully, we did Western Blot experiment through N-terminal located His Tag in proteins, so we managed to show the production of required proteins.

Figure 5

FUNCTIONAL ASSAY

To understand if the proteins we produced are functional, we designed and performed a functional assay. We incubated these two types of bacteria in liquid culture for 13 hours at 37 C; pET45-GadE plasmid containing BL21 bacteria and another BL21 bacteria which contains only pET45-b plasmid for negative control. At 13. our, we added 100 mM IPTG into these liquid cultures. By doing this, we removed the suppression on GadE protein expression. After adding IPTG, we incubated 3 hours more at 37 C.

We prepared LB mediums with different pH values to show produced GadE proteins’ functionality. These LB mediums’ pH values are respectively 7, 5, 3,5; 2,5 and 2. We added thebacteria whichis incubated for 16 hours into LB mediums at the rate of 1:9. This means, for each pH value we added 0.5 ml liquid culture into 4.5 ml LB medium. We also added 1.5 mM Glutamat in each mix and incubated the final mix at 37 C. We made spectrophotometric measurement in 600 nm periodically for the samples that we incubated. Thus we observed how long the bacteria survives in different pH values.

The measurement results are shown below:

Figure 6: 1.5mM Glutamate / 600nm OD / 3h 100mM IPTG

Figure 7

Sequence and Features