Team:uOttawa/Demonstrate

Demonstrate

In summary, our team has created an efficient protocol with the purpose of building Type IIS compatible basic parts and used this protocol to construct a library of Type IIS and RFC 10 compatible plasmids that permitted cloning in both E. coli and S. cerevisiae. Please see below the list of plasmids/plasmid backbones made to date. Please see the Human Practices page for a detailed account of how each plasmid backbone was created.

1. Ade2::EXSP pSB1K3 (RFC10 backbone only) – BBa_K3271000
2. Ade2::EX-RFP-SP pSB1K3 (RFC10 backbone only) – BBa_K3271008
3. Ade2::KanMX-EXSP pSB1K3 (RFC10 backbone only) – BBa_K3271004
4. Ade2::KanMX-EX-RFP-SP pSB1K3 (RFC10 backbone only) – BBa_K3271012
5. His3::EXSP pSB1K3 (RFC10 backbone only) – BBa_K3271002
6. His3::EX-RFP-SP pSB1K3 (RFC10 backbone only) – BBa_K3271010
7. His3::NatMX-EXSP pSB1K3 (RFC10 backbone only) – BBa_K3271007
8. His3::NatMX-EX-RFP-SP pSB1K3 (RFC10 backbone only) – BBa_K3271015
9. Gal4::EXSP pSB1K3 (RFC10 backbone only) – BBa_K3271003
10. Gal4::EX-RFP-SP pSB1K3 (RFC10 backbone only) – BBa_K3271011
11. Gal4::His3-EXSP pSB1K3 (RFC10 backbone only) – BBa_K3271006
12. Gal4::His3-EX-RFP-SP pSB1K3 (RFC10 backbone only) – BBa_K3271016
13. Ade4::EXSP pSB1K3(RFC10 backbone only) – BBa_K3271001
14. Ade4::EX-RFP-SP pSB1K3 (RFC10 backbone only) – BBa_K3271009
15. Ade4::Ura3-EXSP pSB1K3 (RFC10 backbone only) – BBa_K3271005
16. Ade4::Ura3-EX-RFP-SP pSB1K3 (RFC10 backbone only) – BBa_K3271014
17. Ade2::EX-RFP-SP pSB1K00 (RFC10 and Type IIS backbone) – BBa_K3271017
18. Ade2::KanMX-EX-RFP-SP pSB1K00 (RFC10 and Type IIS backbone) – BBa_K3271012
19. His3::EX-RFP-SP pSB1K00 (RFC10 and Type IIS backbone) – BBa_K3271019
20. His3::NatMX-EX-RFP-SP pSB1K00 (RFC10 and Type IIS backbone) – BBa_K3271023
21. Gal4::EX-RFP-SP pSB1K00 (RFC10 and Type IIS backbone) – BBa_K3271020
22. Gal4::His3-EX-RFP-SP pSB1K00 (RFC10 and Type IIS backbone) – BBa_K3271024
23. Ade4::EX-RFP-SP pSB1K00 (RFC10 and Type IIS backbone) – BBa_K3271018
24. Ade4::Ura3-EXSP pSB1K00 (RFC10 and Type IIS backbone) – BBa_K3271022
25. RFP pSB1K00 (RFC10 and Type IIS backbone) --BBa_K3271031

The final step in the creation of the plasmid library consisted of testing and validating that the plasmids can indeed perform homologous recombination in S. cerevisiae.

Testing and validating the plasmid library.

Because the plasmids in the library were made by cloning in E. coli, we had already validated that they are compatible with cloning in E. coli. Next, they had to be validated for cloning in S. cerevisiae. We chose the 4 above underlined plasmids. By confirming that they perform homologous recombination in yeast, we would have also validated all the plasmids in the library because we would have proved that the homology sequences facilitate the process of homologous recombination, whereas the selection markers are functional. Both Setti Belhouari and Victoria Feng validated the plasmid library. Setti Belhouari, who had previous experience cloning in S. cerevisiae, demonstrated that the plasmids, do indeed permit homologous recombination in yeast. Victoria Feng, who had no previous synthetic biology experience, demonstrated that the plasmids indeed facilitate cloning in S. cerevisiae.

Setti PCR amplified a yeGFP cassette (pGPD-yeGFP-tCYC1, BBa_K3271029) flanked by a prefix and a suffix was digested with NotI and directly ligated within the prefix and the suffix of each of the NotI digested plasmids (following the heat-inactivation of NotI endonuclease). The average ligation efficiency of yeGFP into all four plasmids was 36.54 ± 17.06% (see centre-most column in Figure 25). Figure 43 shows the gel electrophoresis of the PmeI digested plasmids from 13 screened colonies following E. coli transformation of aspired ligated Ade4::Ura3-yeGFP in pSB1K3. Presence of yeGFP within the target-loci homologies of the plasmids was further confirmed by PCR using primers complementary to the upstream and downstream homologies as well as primers complementary to the promoter and terminator of yeGFP.

Figure 43.. Identification of yeGFP ligated to Ade4::Ura3 in pSB1K3. A yeGFP amplicon and an Ade4::Ura3 pSB1K3 plasmid were digested with heat-labile NotI and directly ligated then transformed in E. coli and grown on LB kanamycin. Thirteen colonies were screened by PmeI digestion of their miniprep extracted plasmids, which were then gel electrophoresed. The negative control is the PmeI digest of the Ade4::Ura3 in pSB1K3 prior to the introduction of yeGFP. Colonies 1, 10, 11, and 12 contain the desired plasmid whose digestion resulted in bands 3300 bp and 2181 bp long, which correspond to Ade4::Ura3-yeGFP and the pSB1K3 backbone, respectively. Well 8 was a mixture of two colonies, one containing the plasmid with yeGFP and the other not. Identification of Well 7 was impossible due to poor resolution.

The yeGFP cassette in the Ade2, Ade4, and Gal4-targeting plasmids (Ade2::KanMX-yeGFP, Ade4::Ura3-yeGFP, and Gal4::His3-yeGFP) was PCR amplified, then column purified, and then transformed in S. cerevisiae in duplicate, resulting in an average of 18.5 ± 2.1, 122.7 ± 39.5, and 0.7 ± 0.6 colonies per plate, respectively. In a preliminary assay, patches of the Ade2::KanMX-yeGFP transformation were subjected to Alexa 488 protocol, causing an average of 15.0 ± 7.1 % to fluoresce (Figure 44A). A colony PCR was conducted on a seemingly fluorescing colony to ensure the insertion of the yeGFP cassette in the Ade2 locus (Figure 44B).

Figure 44. Preliminary assessment of the insertion of the Ade2::KanMX-yeGFP cassette in W303a. The cassette was PCR amplified from the plasmid similar to the one illustrated in Figure 19. Approximately 780 ng were transformed in strain W303a in duplicate on G418 plates. Ten colonies from both plates were patched on G418 and illuminated using Alexa 488 protocol using BioRad ChemiDocTM MP Imaging System. Two patched colonies, from plate 1, in A are strongly fluorescing. B) A colony PCR was performed on the bottom-left fluorescing patch to detect the insertion of the yeGFP cassette in the Ade2 locus. Primers used amplified the upstream Ade2 homology and the KanMX selection marker, totalling 2452 bp, which was not found in the yeast strain prior to the transformation, or in the zero control.

Victoria repeated the above process independently. She replaced RFP with the yeGFP cassette in the underlined plasmids from the above section. She digested the plasmids using PmeI and gel electrophoresed them, resulting in Figure 45. She then transformed the DNA using the Handbook of Tested and Optimized Synthetic Biology Protocols for Amateurs into S. cerevisiae, and obtained the abundant growth depicted in Figure 46. Therefore, our plasmid backbone library indeed facilitates cloning in S. cerevisiae while remaining compatible with cloning in E. coli via RFC 10 and Type IIS Assembly.

Figure 45. The following plasmids, after the ladder, were digested with PmeI and gelled in this order: His3::NatMX-EXSP pSB1K3, His3::NatMX-EX-yeGFP-SP pSB1K3, Ade2::KanMX-EXSP pSB1K3, Ade2::KanMX-EX-yeGFP-SP pSB1K3, Ade4::Ura3-EXSP pSB1K3, Ade4::Ura3-EX-yeGFP-SP pSB1K3, Gal4::His3-EXSP pSB1K3, Gal4::His3¬-EX-yeGFP-SP pSB1K3.

Figure 46. Victoria Feng, who had no previous synthetic biology experience, used our Handbook of Tested and Optimized Synthetic Biology Protocols for Amateurs as well as our plasmid backbone library to transform yeGFP into S. cerevisiae, resulting in these colony-rich plates.