Part:BBa_K4417013

CuO-RBS-ureEFG-rrnB T1 Terminator TU2

Description

The coding sequence of ureEFG (BBa_K4417010) was cloned into pCT5c (BBa_K4417000) and could be induced by cumate promoter (BBa_K4417007). Besides, this composite contains a strong RBS (BBa_K4417008) and an rrnB T1 terminator (BBa_K4417011). This composite part is the ureEFG gene from Sporosarcina pasteurii.

ureEFG are urease (accessory) proteins, responsible for the activation of the ureABC proenzyme. ureEFG is responsible for the transport and assembly of the nickel II center.

Figure 1: Composite part: CuO-RBS-ureEFG-rrnB T1 Terminator TU2.

Usage and Biology

• This composite part can be used to produce urease accessory proteins.
• The part was synthesized by IDT as a gBlock.

Cloning Strategy

This part was flanked by SapI Type IIS prefix and suffix in order to facilitate sharing of the constructs among the scientific community. In addition, BamHI and SacI sites were used to clone this transcriptional unit into pCT5c plasmid using restriction enzyme digest.

Figure 2: Construct: CuO-RBS-ureABC-rrnB T1 Terminator TU2.

Construct TU2 was ligated with ureEFG (BBa_K4417013) and pCT5c (BBa_K4417000). In Figure 2, the cloned plasmid was checked by diagnostic digest. ureEFG has a smaller size than ureABC, indicated by the bottom band size deviation. Correct band size was observed with 6880bp and 2264bp.

Figure 3: Diagnostic digest of construct TU2. 1: DNA ladder, 2: pCT5c cut with SapI, 3: Construct TU2 cut with SapI (6880bp, 2264bp), 4: Construct TU1 cut with SapI (6880bp, 2757bp).

Construct TU2 was further verified from Sanger sequencing.

Figure 4: Part of the TU2 sequencing results. The top sequence was cloned by Snapgene, and the bottom sequence is our cloned plasmid.

Characterization

SDS PAGE

In order to observe whether the ureEFG was successfully expressed, we analysed our cell pellet using SDS PAGE. The pellet obtained from the 10 mL cultures was then resuspended in Tris Buffer Saline at an OD₆₀₀. Once resuspended, the sample was cell lysed using sonication. Following sonication, the samples were spanned to separate the soluble and insoluble fragments from the whole cell lysate. 60 μL from each sample were obtained and stained with Laemmli reagent.

Figure 5: SDS PAGE of full urease operon in E. coli. All the strains were grown in LB medium; 1: PageRuler Protein Ladder, 2: WT E. coli cell lysate, 3: WT E. coli soluble fragment, 4: SDM1,3 TU1 cell lysate, 5: SDM1,3 TU1 soluble fragment, 6: pCT5c TU1 cell lysate, 7: pCT5c TU1 soluble fragment, 8: SDM1,3 TU2 cell lysate, 9: SDM1,3 TU2 soluble fragment, 10: pCT5c TU2 cell lysate, 11: pCT5c TU2 soluble fragment, 12: pCT5c full urease operon cell lysate, 13: pCT5c full urease operon soluble fragment, 14: pCT5c full urease operon cell lysate, 15: pCT5c full urease operon soluble fragment.

From Figure 5, it could be concluded that the urease was successfully produced since the correct bands are observed in both the soluble and insoluble fragment. ureF was identified at 24.9 kDa.

Reference

1. Zerner, B. “Recent advances in the chemistry of an old enzyme, urease.” Bioorg. Chem. 19 (1991):116-131

2. Krajewska, Barbara. "Ureases I. Functional, catalytic and kinetic properties: A review". Journal of Molecular Catalysis B: Enzymatic 59 no.1-3 (2009):9–21. doi:10.1016/j.molcatb.2009.01.003

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 1085
  Illegal XhoI site found at 1839
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]