Part:BBa_K1758201

His-tagged lac repressor under the control of the T7 promoter

Usage and Biology

Uses T7 promotor and a strong RBS (BBa_K525998) and the lac repressor with a His-Tag. For a higher production of lacI we combine the gene for lacI with T7-RBS (Protein expression can be induced via rhamnose). By adding a His-Tag, lacI can be purified via Ni-NTA columns.

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]

Results

First, we expressed LacI-His and purified the proteins through FPLC from BioRad. For verification of LacI, we ran a SDS-gel (see figure 1).

Figure 1: SDS-PAGE for the identification of LacI.

We cut out the gel band and could identify the band through mass spectrometer measurement with a Mascot score 765.

Further, we established a new assay named "plasmid repressor interaction assay"(PRIA), an in vitro cell-free system based on the binding of a purified repressor protein to purified DNA. In our developed assay, a repressor protein forms a complex with a plasmid containing the corresponding operator sequence. The repressor changes its conformation upon binding of the targeted substrate (further referred to as "analyte") and the bond to the DNA is broken. This disruption will be detected via a loss of a fluorescence signal caused by elution of labeled protein or DNA. As the LacI-lacO system is well characterized, we used it. Read the protocol for PRIA [http://2015.igem.org/Team:Bielefeld-CeBiTec/Protocols#PRIA here].

Figure 2: General workflow of the Plasmid Repressor Interaction Assay

The procedure performed for PRIA was based on his-tagged repressor proteins immobilized on Ni-NTA agarose.(For the general workflow see figure 2.) Herefore, we constructed a device for the expression of his-tagged LacI.

Figure 3: Effect of different salt concentrations on DNA amount in the first elution step. The PRIA was performed with our model system consisting of immobilized LacI. The plasmid contained the lac operator. Agarose gels with samples from washes and elutions of each step of the PRIA after addition of the plasmid. Each PRIA was performed with a buffer containing different amounts of potassium chloride. The only difference between the wash and the elution buffer was the addition of the analyte (in this case IPTG). After the third elution step, the protein bound to the Ni-NTA agarose was eluted with a buffer containing a high concentration of imidazole to confirm the protein's presence at the end of the assay. The Ni-NTA agarose was also applied to the gel to check for remaining plasmid bound to it. The negative control was plasmid applied to Ni-NTA agarose without protein and washed with 250 mM KCl buffer. According to the tests with different salt concentrations in buffer we need to use the buffer with 500 mM salt.

After purification of the protein with the Protino® Ni-TED 1000 Packed Columns Kit from Macherey-Nagel we immobilized it on Ni-NTA agarose in a reaction tube. Plasmid DNA containing the operator site for specific binding of the repressor was added to the Ni-NTA agarose, unbound plasmid was washed out and the remaining plasmid could be eluted by addition of the analyte to the wash buffer. The DNA amount eluted upon addition of the analyte depends strongly on the salt concentration in the buffer used for washing and elution (see figure 3).

We analyzed the released DNA in the supernatant with agarose gel electrophoresis. The DNA amount eluted in the first elution step compared to the total DNA amount bound to the agarose after the forth washing step, is much higher (see figure 4).

Figure 4: Quantification of DNA in different steps of PRIA with PicoGreen assay, n=3. The DNA is mainly eluted after the addition of the analyte in the elution step, but not in the washing steps. This shows that our assay operates as expected.

An explanation for an increased DNA amount in the elution steps could have been dissociation of the protein and the bound DNA from the Ni-NTA agarose. The last step of the assay is elution of the protein from the Ni-NTA agarose with an imidazole buffer to confirm the presence of the protein at the end of the procedure. Our analysis of the samples via SDS-PAGE shows clearly, that there is nearly no loss of protein during the assay and most of the protein can be recovered upon elution with imidazole (see figure 5). On a future test strip the immobilized DNA-protein complex would be provided, so the user just needs to add the potentially contaminated water plus sodium chloride and buffer solution which we would provide as a biosensor kit.

Figure 5: Test for loss of protein in different steps of PRIA.

Moreover, we tested many different conditions to further evaluate the robustness of PRIA. Tap water with the analyte alone could not disrupt the binding between the plasmid and the protein, the major part of plasmid remains bound to the protein. In an agarose gel DNA can be detected in the steps where the protein (and the DNA bound to it) is eluted and in the fraction with the rest of the Ni-NTA agarose, which also still contains protein-DNA complexes. Thus, the user would be required to add a certain amount of salt to the sample. In our assay we mainly applied potassium chloride, but common sodium chloride is suitable for that purpose, too (see figure 8). Nevertheless, the solution should be slightly buffered with TRIS-HCl or sodium/potassium phosphate.

Figure 6: PRIA performed with water instead of buffer

Figure 7: Usage of 500 mM NaCl instead of 500 mM KCl in binding buffer

To further reduce the time for the assay, we also tested whether the complex could be formed prior to addition of the complex to the Ni-NTA agarose. The results were similar to the performance with water instead of buffer (see figure 6 and 7). In this case the bond between repressor and plasmid could not be disrupted at all. In the agarose gel DNA was visible after the elution of the protein with imidazole. This was performed to verify the presence of the protein at the end of the assay. Furthermore, DNA could still be detected on the Ni-NTA agarose (see figure 8).

Figure 8: PRIA with DNA-protein complex added to Ni-NTA agarose

To further analyze our purified LacI, we performed an electrophoretic mobility shift assay (EMSA). The indicated amount of protein was added to each lane. LacI bound specifically to the lac-operator, thereby causing the visible shift in the third lane (see figure 9).

Figure 9: Interaction study of LacI-lacO through electrophoretic mobility shift assay.