Part:BBa_K1492001

Clickable Outer Membrane Protein y

INPNC-MCS with TAG and HA-tag.
See BBa_K811005 for more info. This Biobrick has a TAG codon at C Terminus for the introduction of any Non Natural Amino acid. The biobrick also has a HA-tag for detection on the membrane protein in the cell membrane.

Usage and Biology

In 2012, the iGEM team of Penn, University of Pennsylvania, created a generalized BioBrick surface display platform: the Ice Nucleation Protein N-, C-termini only (INPNC), BioBrick part: BBa_K811005. INPNC is a truncated Ice Nucleation Protein (INP, a protein which causes ice nucleation and formation but which is also used for its surface display properties). However, INP consists of a C-terminal region that is positioned on the outer membrane, as well as a central 8, 16 or 48 amino acid motif that is responsible for INP’s ice nucleation properties. These central amino acids motifs are not essential for INP’s surface displaying properties. Therefore INP was truncated, retaining only the N (179 aa) and C termini (49 aa) to create INPNC. The C-terminal domain is displayed on the cell surface, while the N-terminal domain remains in the outer membrane.

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
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 72
  Illegal NgoMIV site found at 405
  Illegal AgeI site found at 823
  
• 1000
  COMPATIBLE WITH RFC[1000]

Gene Design:

The protein has a TAG codon and HA-tag added at the C-terminus of the protein. The TAG codon is for the incorporation of a Non Natural Amino acid. With the proper tRNA synthetase any Non Natural Amino Acid can be incorporated into the protein and displayed on the membrane. The HA-tag was used for characterization of the protein. Both modifications have been made by using an overhang primer. After the modification the protein has been renamed to Clickable Outer Membrane Protein y (COMPy).

We as team TU Eindhoven 2014 state that we have improved the functionality and “ease of use” of BioBrick part: BBa_K811005 [1]. The functionality has been improved by adding the TAG. This allows the incorporation of any non-natural amino acid. The “ease of use” has been improved by the introduction of a HA-tag to the protein. This tag makes it a lot easier to screen whether or not the protein is expressed using fluorescent anti-HA antibodies.

Figure 1: Gene design of COMPy

Characterization:

Team TU Eindhoven 2014 tried to create a membrane anchor to which molecules could covalently bind. With this protein it is possible to bind anything to the membrane by using a bio-orthogonal “click” reaction. To test the functionality of the protein several assays were done.

For all the assays we used the following vectors pET29a(+) COMPy (Membrane Protein) and pEVOL-PylT-2xTyrRS. (tRNA, tRNA synthetase). Both vectors were transformed into BL21(DE3) strain. Colonies of this transformation were grown LB. After culturing glycerol stocks were made. All the assays were done by culturing the bacteria from this glycerol stock. Check our Protocol Page for all the protocols of TU Eindhoven 2014. For all the protocols of TU Eindhoven 2014 see: http://2014.igem.org/Team:TU_Eindhoven/Protocols

Antibody confirmation:

First assay done was to confirm that the protein was expressed on the membrane. This was done by adding an anti-HA antibody labeled with TAMRA dye to bacteria in solution that had been expressing COMPy. The bacteria were then analyzed with FACS. Results show that COMPy is expressed on the membrane of the bacteria (Figure 2). Though it remains a small increase, reasons for this can be that the bacteria are having trouble expression the protein itself or the non-natural amino acid causes problems for expression. http://2014.igem.org/Team:TU_Eindhoven/Protocols -> Antibody Labeling

Figure 2: COMPy labeled with Anti-HA antibody

DBCO-PEG4-TAMRA Confirmation:

Now that it is proven that the protein is expressed on the membrane the next step can be taken. This assay is to see whether or not the non-natural amino acid is being incorporated into COMPy. For labeling DBCO-PEG4-TAMRA was used. If the non-natural amino acid is present in the protein then DBCO-PEG4-TAMRA should be measured after washing steps. DBCO-PEG4-TAMRA was added in two different concentrations, in excess, to bacteria solution after expression of COMPy was induced. The bacteria were then analyzed with FACS. Data clearly shows that DBCO-PEG4-TAMRA is still on the membrane after washing steps (Figure 3). Therefore the DBCO of the dye is clicked to the azide, in other words: the dye has covalently bound to the protein. The fact that there is also a lot of signal at a lower fluorescence (around 10²) goes to show that there is a lot of variance between bacteria expressing COMPy. A peak at high fluorescence is what is expected when the bacteria produce the protein well. For protocol see: http://2014.igem.org/Team:TU_Eindhoven/Protocols -> FACS for sorting with DBCO-TAMRA

Figure 3: COMPy labeled with DBCO-PEG4-TAMRA

Influence Non Natural Amino Acid on the expression of COMPy:

To be able to determine the influence of the Non Natural amino acid on COMPy a small modification had to be done to the protein. The TAG codon has been mutated into a TTG codon, coding for a serine. The mutated protein is called COMPy noTAG. After mutation the new plasmid has been transformed into a BL21 strain and cultured. The original COMPx was also cultured. Protein expression has been induced and the pAzF amino acid has been given to both cultures. After expression both types were labeled with Anti-HA antibody in two concentrations and have been analyzed with the FACS. For protocol see: http://2014.igem.org/Team:TU_Eindhoven/Protocols -> Antibody Labeling

Figure 4: COMPy labeled with Anti-HA antibody

Figure 5: COMPy noTAG labeled with Anti-HA antibody

The expression of COMPy is lower than the expression of COMPy noTAG, in Figure 5 the peaks are at a higher fluorescence than the peaks in Figure 4. Furthermore this data shows that COMPy and COMPy noTAG do not express well in general. Even for COMPy noTAG, which does not use the non-natural Amino acid, the expression is not that good.