Plasmid

Part:BBa_K2141000:Experience

Designed by: Altug Uludag, Nedim Haciosmanoglu, Galip Can Guclu, Yagmur Guneri, Melis Akgun   Group: iGEM16_Istanbul_Tech   (2016-10-04)


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Team Oxford 2019

We used this part for protein production in E. coli. pET28A was a very useful vector, as its T7 promoter provided strong protein expression, and kanamycin resistance allowed for easy antibiotic selection, without interfering with the chloramphenicol resistance used in our BL21(DE3)-RIPL E. coli expression strain. Kanamycin concentration of 50 μg/ml were used with this plasmid on both low-salt Lysogeny Broth (LB) agar and in liquid LB broth. Following transformation, we allowed SOC outgrowth for 45 minutes. This was likely more than enough time, and we had no issues with cells which transformed but weren't given sufficient time to express kanamycin resistance.

Below we show two protein purification gels. One sample of Ni-NTA purified SpyTag-mClover3-6His from the pET28A system, indicating high protein production, while another is shown with CD27L endolysin production:

Figure 1: SDS-PAGE Ni-NTA Affinity Purification of SpyTag-mClover3-6His
Figure 2: SDS-PAGE Ni-NTA Affinity Purification of 6His-SpyTag-CD27L























Additional Information

Team: iGEM-IISERPUNE-2020

  • Pet28a(+) is widely used for expression purpose. But unnecessary components often interfere in cloning strategies. So we altered the part into a modified version that satisfies RFC10 criteria, compatible with Gibson's cloning and all the unnecessary components removed: BBa_K358500.


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