Team:NYMU-Taipei/Cloning

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Protein Expression

As previous research showed, tuberculosis and lung cancer patients’ bodies give off heptanal1, and mOR103-15 mouse olfactory receptor can bind with it2. Thus, we used mOR103-15 to exemplify Volatile Organic Compounds (VOCs) biomarkers for early disease screening. In our experiments, mOR103-15 was bought from Addgene, and

  1. PCR cloned it into the vector BBa_J364007, to enhance intracellular expression of mouse olfactory receptor mOR103-15 in E. coli.
  2. directly used the plasmid bought from Addgene to express mouse olfactory receptor mOR103-15 in E. coli.
  3. PCR cloned it into the vector BBa_J36850, to express mouse olfactory receptor mOR103-15 on the E. coli outer membrane.

mOR103-15

MOR103-15 was bought from Addgene. We first used BamHI & PstI directly cleaving the product we received to ensure it was correct. The two segments were supposed to be 559bp and 3442bp, respectively, which was confirmed by the result of electrophoresis (Fig 2).

Fig 1. Plasmid map of PI7-ISH, which was bought from Addgene.
Fig 2. Comfirmation of PI7-ISH.

Then we extracted plasmid by Geneaid’s Presto™ Mini Plasmid Kit.

Intracellular Expression

Cloning into the Vector BBa_J364007

We underwent PCR [100ng plasmid, 2μl primers solution (5μl each/90μl H2O), 2μl each dNTP, 2μl reaction buffer, 1.2μl MgSO4, 1μl KOD+, sterilized H2O added to 20μl]. Then, we used BglII & HindIII to cleave the insert mOR103-15, while used BamHI & PstI to cleave the vector BBa_J364007. Finally, we ligated both by New England Biolabs’ T4 DNA Ligase, and transformed into E.coli DH5-alpha.

Fig 3. Plasmid map of BBa_K3268000/in_mOR103-15.

Transformation was performed in six different tubes. To confirm which construct was correct, we used 6 sets of restriction enzymes to cleave the construct and ran gel electrophoresis: (1) EcoRI / BamHI, which would cleave the plasmid into 436bp and 2821bp segments; (2) XbaI / BamHI, which would cleave the plasmid into 512bp and 2736bp segments; (3) XbaI / HindIII, which would cleave the plasmid into 1052bp and 2205bp segments; (4) NcoI, which would cleave the plasmid into 1313bp and 1944bp segments; (5) NotI, which would cleave the plasmid into 1211bp and 2046bp segments; (6) XhoI, which would cleave the plasmid into 892bp and 2365bp segments.

Fig 4. Construct confirmation by 6 sets of restriction enzymes.

From the results, we concluded that the fourth construct met our needs.


To confirm that E.coli would express our desired protein, mOR103-15, we subsequently lysed the cells (procedure is written on the Immobilization page). Then, centrifuged at 14-16,000 x g for 5-10 mins, and saved supernatant; mixed 15μl each sample with 5μl 4X loading dye, and boiled at 70-80C for 10 mins. Finally, we ran a SDS-PAGE gel3,4 (for separating gel, we used 12% bis/acrylamide, 0.1% SDS, 375mM Tris-HCl at pH8.8, 50μl 10% APS, 5μl TEMED, and water added to 10ml; for stacking gel, we used 5% bis/acrylamide, 0.1% SDS, 125mM Tris-HCl at pH6.8, 30μl 10% APS, 5μl TEMED, water added to 5ml), and used Bioman Scientific's RAPID stain to stain the gel.

We compared moR103-15 protein to some fluorescence proteins and lysozyme, which we used to lyse the cell (molar weight: mCherry is 26.7kDa5, eforCP is 25.7kDa6, AmilCP is 25.0kDa7, sfGFP is 26.8kDa8, mOR103-15 is 36.2kDa9, lysozyme is 14.3kDa10). The result implied that E.coli can express the protein we wanted. The result implies that E.coli can express the protein we wanted.

Fig 5. Expression of mOR103-15 compared to fluorescent proteins and lysozyme.

Directly Using Addgene's Plasmid

We directly transformed the plasmid bought from Addgene into E. coli BL21(DE3) to express mOR103-15. As mOR103-15 is controlled by T7 promoter on the plasmid, we chose E. coli BL21(DE3), who has T7 RNA polymerase to activate the promoter.

After transformation, we made overnight liquid cultures. Then, we conducted IPTG induction so that T7 RNA polymerase would be expressed. The materials we used include: (1) overnight culture, (2) LB broth medium, (3) 1M IPTG. The following is the procedure:

  1. Dilute 1:100 overnight culture, since the total volume is 50mL.
  2. Take 50μl cell culture, and incubate at 37ºC for 3.5 hr. After that, measure OD. In our experiment, it is 1.040, which is appropriate.
  3. Add 25μl IPTG to 50mL cell culture, such that IPTG concentration is 0.5mM. Mix well, and incubate for 3-4 hr.
  4. Prepare uninduced sample before IPTG added, with 1mL culture spin down, and get pellet. Store at -20ºC.
  5. Induce sample. Transfer 1mL to the eppendorf, spin down, and get pellet. Store at -20ºC.

Subsequently, we used the rest 48mL cell culture to conduct cell lysis (procedure is written on the Immobilization page).

Surface Display

Cloning into the Vector BBa_J36850

In order to display mor103-15 on the surface of cell, we found that the Lpp′OmpA(46-159) hybrid protein can serve as an efficient targeting vehicle for localizing a variety soluble proteins onto the E. coli surface11,12. We got the part BBa_J36850 which is combined with a lac promoter and strong ribosome binding site for lac-inducible expression of the fusion protein of Lpp signal peptide, OmpA aa46-159, and streptavidin wild-type + His6 tag from iGEM kit 2019.


We underwent PCR [100ng plasmid, 2μl primers solution (5μl each/90μl H2O), 2μl each dNTP, 2μl reaction buffer, 1.2μl MgSO4, 1μl KOD+, sterilized H2O added to 20μl]. Then, we used BglII & HindIII to cleave the plasmid for the purpose of removing the streptavidin wild-type, and we also used used BglII & HindIII to cleave the insert mOR103-15. Finally, we ligated both by New England Biolabs’ T4 DNA Ligase, and transformed into E.coli DH5-alpha.

Fig 6. Plasmid map of BBa_K3268001/out_mOR103-15

Transformation was performed in 8 different tubes. To confirm which construct was correct, we used 2 sets of restriction enzymes to cleave the construct and ran gel electrophoresis: (1) BglII / HindIII, which would cleave the plasmid into 1140bp and 2605bp segments; (2) EcoRI / BamHI, which would cleave the plasmid into 984bp and 2761bp segments.

Fig 7. Construct confirmation by 2 sets of restriction enzymes.

From the results, we concluded that the first, fourth, sixth, and seventh construct were correct. To display our target protein on the outer membrane, we used IPTG to induce. We chose the fourth, sixth, seventh, and eighth construct (as control group) to be induced13.

References

  1. Mellors, T. R. et al. Identification of Mycobacterium tuberculosis using volatile biomarkers in culture and exhaled breath. J Breath Res 13, 016004, doi:10.1088/1752-7163/aacd18 (2018).
  2. Wang, X. et al. Peptide surfactants for cell-free production of functional G protein-coupled receptors. Proceedings of the National Academy of Sciences 108, 9049-9054, doi:10.1073/pnas.1018185108 (2011).
  3. Brunelle, J. L. & Green, R. One-dimensional SDS-polyacrylamide gel electrophoresis (1D SDS-PAGE). Methods Enzymol 541, 151-159, doi:10.1016/b978-0-12-420119-4.00012-4 (2014).
  4. Gallagher, S. R. One-dimensional SDS gel electrophoresis of proteins. Curr Protoc Protein Sci Chapter 10, Unit 10.11.11-44, doi:10.1002/0471140864.ps1001s68 (2012).
  5. mCherry, <https://www.fpbase.org/protein/mcherry/>
  6. eforCP, <https://www.fpbase.org/protein/eforcp/>
  7. amilCP, <https://www.fpbase.org/protein/amilcp/>
  8. Superfolder GFP, <https://www.fpbase.org/protein/superfolder-gfp/>
  9. National Center for Biotechnology Information. olfactory receptor 2 [Mus musculus], <https://www.ncbi.nlm.nih.gov/protein/NP_035113.1>
  10. Sigma-Aldrich Co. LLC. Lysozyme from chicken egg white, <https://www.sigmaaldrich.com/catalog/product/sigma/l7651?lang=en&region=TW> (2018).
  11. Stathopoulos, C., Georgiou, G. & Earhart, C. F. Characterization of Escherichia coli expressing an Lpp’OmpA(46-159)-PhoA fusion protein localized in the outer membrane. Applied Microbiology and Biotechnology 45, 112, doi:10.1007/s002530050657 (1996).
  12. Verhoeven, G. S., Alexeeva, S., Dogterom, M. & Blaauwen, T. d. Differential Bacterial Surface Display of Peptides by the Transmembrane Domain of OmpA. PLOS ONE 4, e6739, doi:10.1371/journal.pone.0006739 (2009).
  13. Biologics International Corp. IPTG Induction Protocol, <https://www.biologicscorp.com/blog/iptg-induction-protocol/#.XaAnGkYzY2x>