Methods

Correct 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 suite. Further information on PyMOL can be found here.

• Structure check of HER2: The structure of HER2 with the PDB ID: 3MZW was validated with the use of the program PROCHECK.
• 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 BLAST search was performed with the sequence of HER2 (3MZW_HER2-receptor.fasta) followed by a manual selection of 11 sequences from different organisms. Then a multiple sequence alignment was performed using CLUSTAL 2.1. The obtained alignment could then be used for conservation calculation on the ConSurf Server.

  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

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.

Conversion of BACTH into an iGEM standard and analysis of function

Set up of flow system

References

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2. The PyMOL Molecular Graphics System, Version 1.7.4 Schrödinger, LLC.
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