Team:TU Dresden/Project/Methods


Methods

Here you can find a small summary of the methods we used in each subproject:

Folding study of target protein

  1. Primer design for adding flanking regions which contained restriction sites for adding HER2 to pET28a which contains His-tag.
  2. PCR to add the above mentioned flanking regions to HER2.
  3. A restriction digestion was done on pET28a plasmid with the enzymes NheI-HF and NotI-HF simultaneously.
  4. The PCR product obtained was also subjected to restriction digestion by the same enzymes.
  5. Both the digests were run on a gel to confirm restriction digestion and a gel extraction was performed to get them back.
  6. Now both the products were pooled together in the molar ratio of 1 part of pET28a to 2.5 parts of HER2 and an overnight ligation was performed with T4 ligase. the concentration was determined using a nanodrop.
  7. These ligated products were then electroporated into Escherichia coli GB05 which were streaked onto kanamycin plates for selection.
  8. These plates were checked for colonies and overnight cultures were setup from selected colonies.
  9. A plasmid prep was performed on these o\n cultures to extract the pET28a-HER2 plasmid, following which a restriction digestion was performed with the enzymes NheI-HF and NotI-HF and run on a gel to confirm if the ligation was correct.
  10. After this the plasmids extracted were sent for sequencing to double check if the ligated products were correct.
  11. After finding that the sequences were correct, the plasmid pET28a-HER2 was electroporated into E. coli BL21 for protein expression.
  12. A plasmid prep, a restriction digestion and a gel electrophoresis were done in tandem to confirm that the plasmids electroporated were correct.
  13. After this 4 litre cultures were started from which protein extraction was performed.
  14. The proteins extracted were subjected to affinity chromatography using a column with Ni which was specific to His-tag.
  15. The elution form the affinity column was subjected to size exclusion chromatography which gave 4 distinct peaks of which only three were selected and one was excluded as it was the peak for imidazole.
  16. The elutes of the three peaks were subjected to CD (circular dichorism) spectroscopy which gave similar results to all three (majority alpha helices).
  17. The deconvolution results were then compared with that from PDB to analyse if the protein has been folded correctly.
  18. In addition a Blue Native PAGE was performed to detect if the different peaks were actually multimers of the same protein.

Structure analysis of our targets and their interactions

For the following analysis. All molecule pictures and the structure video were made using the PyMOL [2] suite. Further information on PyMOL can be found here.

For structure analysis the protein data base structure with the PDB ID: 3MZW was used [1].

  • Structure check of HER2: The structure of HER2 with the PDB ID: 3MZW was validated with the use of the program PROCHECK [3,4].
  • Calculation of intefacial residues of HER2 and its bound affibody: In order to define the interfacial residues of HER2 and its affibody the python script InterfaceResidues.py was used within PyMOL.
  • Calculation of electrostatic interactions in the interface: The electrostatic interactions in the interface were first defined in PyMOL using the preselected interface atoms, atom types and maximum distances. For explicit calculation of hydrogen bonds the Python script list_hbonds.py was used in PyMOL used with a distance cutoff of 3.2 Angsrom and an angle cutoff of 55 degrees. The script automatically adds a requirement that atoms in the selection must be either of the elements N or O.
  • Conservation study of HER2: A search for similar sequences of the sequence of HER2 (3MZW_HER2-receptor.fasta) was performed using the Basic Local Alignment Search Tool (BLAST) [5, 6] followed by a manual selection of 11 sequences from different organisms. Then a multiple sequence alignment was performed using CLUSTAL 2.1 [7]. The obtained alignment could then be used for conservation calculation on the ConSurf Server [8, 9, 10, 11].

    In order to obtain a structure color coded by conservation the new (changed) PDB file obtained from the conservation calculation was loaded into PyMOL together with the Python script consurf_HER2.py.

  • Visualization of the B-factor for the affibody ZHER2: For obtaining a structure color coded by B-factor the Python script color_b.py was used in PyMOL.

Investigation of P3 threshold for E. coli resistance

Transformation of E. coli with the construct

  1. Resuspension of synthesized plasmids T25, T18, ZHER2, LZ-T18, LZ-T25, HER2 and P3.
  2. Resuspension of iGEM plasmids containing RBS (BBa_E0020), CFP (BBa_E0020), and pLac (BBa_K611025).
  3. Transformation of the 10 plasmids into E. coli GB05.
  4. Plating of transfected cells on Cm and Kan plates for synthesized and iGEM plasmids respectively.
  5. Plasmid preparation for the 10 plasmids.
  6. Nanodrop measurement of plasmid prep.
  7. Analytical digest of plasmid prep with NotI.
  8. Digest of P3 and CFP with XbaI and PstI-HF → lin P3, lin CFP.
  9. Digest of pLac and RBS with SpeI and PstI-HF → lin pLac, lin RBS.
  10. Dephosphorylation of pLac and RBS.
  11. Gel-purification of lin P3, lin CFP, lin pLac and lin RBS.
  12. Nanodrop measurement of lin P3, lin CFP, lin pLac and lin RBS.
  13. Ligation of pLac with P3 → (pLac + P3) and RBS with CFP → (RBS + CFP).
  14. Transformation of (pLac + P3) and (RBS + CFP) into E. coli GB05.
  15. Plasmid preparation of (pLac + P3) and (RBS + CFP).
  16. Nanodrop measurement of (pLac + P3) and (RBS + CFP).
  17. Digest of (pLac + P3) with SpeI and Pst-HF → lin (pLac + P3).
  18. Digest of (RBS + CFP) with XbaI and PstI-HF → lin (RBS + CFP).
  19. Dephosphorylation of lin (pLac + P3).
  20. Gel-purification of lin (RBS + CFP) and lin (pLac + P3).
  21. Nanodrop measurement of lin (RBS + CFP) and lin (pLac + P3).
  22. Ligation of lin (RBS + CFP) with lin (pLac + P3) → final construct.
  23. Transformation of final construct into E. coli ER2738.
  24. Medium
    • Lysogeny broth:
      • 10 g L-1 peptone
      • 5 g L-1 yeast extract
      • 10 g -1 sodium chloride
    • Optional:
      • 10 g L-1 agar-agar
      • 25 mg L-1 chloramphenicol
      • 40 mg L-1 X-Gal
  25. Measurement of the expression of P3 in E. coli co-expressed with CFP.

Analysis of the plasmid stability

  1. The plasmid stability was analyzed by using two LB agar plates with and without antibiotics.
  2. The samples were diluted and plated overnight.

Analysis of the phage infection

  1. A 10-5 dilution from the plasmid stability was used to analyze whether phages are washed away during the cultivation.
  2. The samples were plated on a plate with chloramphenicol and X-Gal.

Conversion of BACTH into an iGEM standard and analysis of function

Fusion ligation of T18, LZT18 and T25, LZT25

  1. Restriction digestion of T18 and T25 using the enzymes.
  2. Restriction digestion of LZT18 and LZT25.
  3. Purification of restriction digests using gel electrophoresis.
  4. Ligation of T18 with LZT18 vector and T25 with LZT25 vector respectively.
  5. Transformation into E. coli GB05 strain and plate in agar with kanamycin.

Ligation of fusion products, T18-LZT18 and T25-LZT25

  1. Restriction digestion of T25-LZT25 fusion product with EcoRI and SpeI and T18-LZT18 fusion product with EcoRI and XbaI.
  2. Purification of restriction digests using gel electrophoresis.
  3. Ligation of T25-LZT25 cassette with T18-LZT18 vector. For simplicity reasons let us call this T18 T25 product.
  4. Transformation into E. coli GB05 strain and plate agar with kanamycin.

Ligation with lacZ

  1. Restriction digestion of lacZ using EcoRI and XbaI.
  2. Restriction digestion of T18-T25 product using EcoRI and SpeI.
  3. Purification of restriction digests using gel electrophoresis.
  4. Ligation of lacZ cassette with T18-T25 product.
  5. Transformation into E. coli cya- strain and plate on X-Gal plates.
  6. Observe blue colonies, which contain successful ligation product.

Set up of flow system

For this subproject, two devices were used, which are summarized in the following table:

Device Manufacturer Type series
Fluorescence spectrometer Hitach F-4500
Bioreactor Applikon 1 L

Continuous stirred-tank cultivation

  1. The bioreactor (1 L Applikon) was filled with minimal medium previouly set to a cell density of OD600 of 0.04.
  2. The bioreactor was inoculated with stirred and aerated medium.
  3. The culture was grown until a OD600 of 0.2 was reached.
  4. The continuous cultivation was started (figure 1).
  5. The medium is continuously removed and transported to the lagoon.
  6. Next isopropyl β-D-1-thiogalactopyranoside (IPTG) was added to the feed entering the lagoon.
  7. After 3 hours a sample from the lagoon's outlet was taken and analyzed.
Figure 1 - Setup of the continuous cultivation with lagoon an IPTG-pump.

References

  1. Eigenbrot, C., Ultsch, M., Dubnovitsky, A., Abrahmsén, L., Härd, T. (2010). Structural basis for high-affinity HER2 receptor binding by an engineered protein. Proceedings of the National Academy of Sciences, 107(34), 15039-15044.
  2. The PyMOL Molecular Graphics System, Version 1.7.4 Schrödinger, LLC.
  3. Laskowski, R. A., MacArthur, M. W., Moss, D. S., Thornton, J. M. (1993). PROCHECK - a program to check the stereochemical quality of protein structures. Journal of Applied Crystallography, 26, 283-291.
  4. Laskowski, R. A., Rullmannn, J. A., MacArthur, M. W., Kaptein, R., Thornton, J. M. (1996). AQUA and PROCHECK-NMR: programs for checking the quality of protein structures solved by NMR. Journal of Biomolecular NMR, 8, 477-486.
  5. Goujon, M., McWilliam, H., Li, W., Valentin, F., Squizzato, S., Paern, J., Lopez, R. (2010). A new bioinformatics analysis tools framework at EMBL-EBI. Nucleic acids research, 38 Suppl: W695-9 DOI:10.1093/nar/gkq313.
  6. Altschul, S.F., Gish, W., Miller, W., Myers, E. W., Lipman, D.J. (1990). Basic local alignment search tool. Journal of Molecular Biology, 215, 403-410.
  7. Sievers, F., Wilm, A., Dineen, D. G., Gibson, T. J., Karplus, K., Li, W., Lopez, R., McWilliam, H., Remmert, M., Söding, J., Thompson, J. D., Higgins, D. G. (2011). Fast, scalable generation of high-quality protein multiple sequence alignments using Clustal Omega. Molecular Systems Biology, 7 Article number: 539 doi:10.1038/msb.2011.75.
  8. Celniker, G., Nimrod, G., Ashkenazy, H., Glaser, F., Martz, E., Mayrose, I., Pupko, T., Ben-Tal, N. (2013). ConSurf: using evolutionary data to raise testable hypotheses about protein function. Israel Journal of Chemistry, doi: 10.1002/ijch.201200096
  9. Ashkenazy, H., Erez, E., Martz, E., Pupko, T., Ben-Tal, N. (2010). ConSurf 2010: calculating evolutionary conservation in sequence and structure of proteins and nucleic acids. Nucleic Acids Research, doi: 10.1093/nar/gkq399.
  10. Landau, M., Mayrose, I., Rosenberg, Y., Glaser, F., Martz, E., Pupko, T., Ben-Tal, N. (2005). ConSurf 2005: the projection of evolutionary conservation scores of residues on protein structures. Nucleic Acids Research, 33:W299-W302.
  11. Glaser, F., Pupko, T., Paz, I., Bell, R.E., Bechor, D., Martz, E., Ben-Tal, N. (2003). ConSurf: identification of functional regions in proteins by surface-mapping of phylogenetic information. Bioinformatics, 19, 163-164.