Coding

Part:BBa_K1151000

Designed by: Emanuele Rizzo   Group: iGEM13_UniSalento_Lecce   (2013-09-21)
Revision as of 21:37, 27 September 2013 by LeleBiotec (Talk | contribs) (ICP-AES assay)

Nickel-responsive pleiotropic regulator (HpNikR)

The HpNikR protein is a pleiotropic regulator from Helicobacter pylori. In presence of nickel it can acts as an activator or a repressor depending of the specific promoter that contains its operator site. It consists of two dimeric DNA binding domains separated by a tetrameric regulatory domain that binds nickel. This domain corresponds to the C-terminal regulatory domain which contains four nickel binding sites at the tetramer interface. Binding nickel, then a conformational change allows it to activate or repress trascription.


Usage and Biology

                                       Cattura.jpg                                           

Figure 1: HpNikR nickel-binding domain (tetramer).



Cattura1.jpg

Figure 2: Proteic sequence analysis with Uniprot.


NikR expression using BL21 (DE3) cells

First we made ​​competent BL21 cells and we transformed it with the plasmid containing NikR. We then proceeded with the normal protocol of induction with IPTG.

                                                Rit.jpg

Figure 3: PAGE of the first induction.

Cytosol/membrane separation by Zerial method

To confirm that NikR is a cytosolic protein (not expressed in multivesicular bodies, and then in membrane) we performed a separation membrane-cytosol (Zerial method) (sample: 2-hours induced cells).

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Figure 4: PAGE of the separation.

NikR purification by Ni-NTA resin

NikR (sample: 2-hours induced cells) can be purified by Ni-NTA resin, which has a high affinity for histidine residues.

41.jpg

Figure 5: Purification result (Eluate 1: 0,82 ug/ul).

NikR incubation with nickel sulfate and Sephadex molecular exclusion chromatography

We therefore decided to study the NikR protein: first, we focused on its characteristic to bind nickel. We have developed a protocol of incubation of the protein with nickel sulfate (stock: 10 ug/ul), in presence of an Incubation buffer (20 mM Tris pH 7,6, 100 mM NaCl). These are the samples we tested (each has a final volume of 100 ul):

1. 1,2 ul (1 ug) NikR + 0,3 ul Nickel sulfate + 98,5 ul Incubation buffer

2. 6,1 ul (5 ug) NikR + 1,5 ul Nickel sulfate + 92,4 ul Incubation buffer

3. 12,2 ul (10 ug) NikR + 3 ul Nickel sulfate + 84,8 ul Incubation buffer

The samples were put on wheel at 4°C overnight.

In order to eliminate the nickel eccess we proceeded with a molecular exclusion chromatography on Sephadex G-25 resin.

Resina.jpg

Figure 6: Sephadex columns.

In this way the protein with the nickel-binding sites saturated will be released first from the column, collecting the eluate easily. The nickel in excess will remain trapped in the pores of the resin.

ICP-AES assay

Inductively-Coupled Plasma Atomic Emission Spectroscopy (ICP-AES) is a type of emission spectroscopy that uses the inductively coupled plasma to produce excited atoms and ions that emit electromagnetic radiation at wavelengths characteristic of a particular element. The intensity of this emission is indicative of the concentration of the element within the sample. These are the samples that we have analyzed and the results:

1. 100 ul Incubation buffer

2. 1,5 ul Nickel sulfate + 98,5 ul Incubation buffer

3. 1,2 ul (1 ug) NikR + 0,3 ul Nickel sulfate + 98,5 ul Incubation buffer

4. 6,1 ul (5 ug) NikR + 1,5 ul Nickel sulfate + 92,4 ul Incubation buffer

5. 12,2 ul (10 ug) NikR + 3 ul Nickel sulfate + 84,8 ul Incubation buffer

6. Sephadex resin after incubation with sample n.4



Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


[edit]
Categories
Parameters
n/aNickel-responsive pleiotropic regulator (HpNikR)