Part:BBa_K4417016

CuO-RBS-ureABC-rrnB T1 Terminator TU1B

Description

The coding sequence of ureABC with additional BsaI sites (BBa_K4417015) 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 ureABC gene from Sporosarcina pasteurii (BBa_K4417009). This enzyme contains three urease subunits and is able to catalyze the breakdown of urea into ammonia and CO2. UreA, ureB, and ureC have α, ß, and γ active sites, respectively, which work in coordination with structural change to break down urea. The part is basically the same as BBa_K4417009 with additional BsaI sites for Level 0s cloning.

Figure 1: Composite part: CuO-RBS-ureABC-rrnB T1 Terminator TU1B.

Usage and Biology

• This composite part can be used to produce urease structural complex ureABC.
• This composite part can be used to clone urease accessory proteins (BBa_K4417016) using Golden Gate Assembly.
• 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 part into pCT5c plasmid using restriction enzyme digest. Although BsaI sites were not compatible with the Type IIS standard, two BsaI sites were included for further Level 0s cloning.

Figure 2:Construct: CuO-RBS-ureABC-rrnB T1 Terminator TU1B.

Construct TUM was ligated with ureABC with BsaI sites (BBa_K4417015) and pCT5c (BBa_K4417000). In Figure 3, the cloned plasmid was checked by diagnostic digest. Correct band size was observed of 5803bp, 2608bp, and 1077bp.

Figure 3:Diagnostic digest of construct TUM. 1: 1: DNA ladder, 2: Construct 2 uncut, 3: Construct 2 cut with SapI (6880bp, 2264bp), 4: Construct 1 uncut, 5: Construct 1 cut with SapI (6880bp, 2757bp), 6: Construct 1 cut with SapI (6880bp, 2757bp), 7: Construct TUM uncut, 8: Construct TUM cut with SapI and BsaI (5803bp, 2608bp, 1077bp). 9: Construct TUM uncut, 10: Construct TUM cut with SapI and BsaI (5803bp, 2608bp, 1077bp). All the TUM was in SDM1,3 plasmid with one more BsaI to be mutated.

Construct TUM was further verified from Sanger sequencing.

Figure 4:Part of the TUM sequencing results. The top sequence was cloned by Snapgene, and the bottom sequence is our cloned plasmid. The sequence was shown around the BsaI region.

Characterization

SDS PAGE

In order to observe whether the ureABC 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. ureC was identified at 61.5 kDa.

Urease Assay

We evaluated the activity of urease activity via a calorimetric pH assay. Urease protein catalyzes urea degradation into two ammonia molecules and one carbon dioxide, resulting in the net pH increase of the substrate. The urease activity assay measures precisely this apparent change in acidity. We suspend cell culture in a Tris medium to limit its growth, including phenol red as a pH indicator. As pH increases, phenol red turns redder. Therefore, we can characterize how basic the medium turns, by measuring changes in absorbance of red light (OD₅₆₂). To ensure that these changes are driven by urease activity and not by increase in cell growth, we must correct for any possible cell growth by measuring at OD₇₀₀ as well.

By observing differences between the OD₅₆₂/OD₇₀₀ for transformed and wild type cells, we are able to confirm different changes in pH and hence urease activity.

Figure 6: Urease activity assay of WT E. coli and transformed TU1.

Biocementation

Induced engineered bacteria should precipitate significantly more calcium carbonate in calcium rich environments than wild type bacteria. The first place where this can be observed is on the cell colony morphology. Our engineered bacteria produce crystal-like colonies (on calcium rich LB plates) which points to increased CaCO₃ precipitation caused by our genetic constructs.

Figure 7: Precipitation on calcium rich agar plates by TU1.

The dried aggregate of transformed bacteria is pale and matte compared with the aggregate of wild type bacteria, which is dark brown and has a glass-like surface. This is an indication of enhanced calcium carbonate precipitation.

Figure 8: Dry aggregates from biocementation. TU1B was the same as TU1, except additional BsaI sites.

We measured the dry weight of aggregates formed by the engineered and wild type cells in a calcium and urea rich medium. There is a statically significant (p<0.05) increase in weight between the engineered cells (TU1B, CA17) and the wild type (DH5-α). The increase in dry weight of the aggregate also points to enhanced calcium carbonate precipitation of the engineered cells.

Figure 9: Dry weight of aggregates from biocementation. TU1B was the same as TU1, except additional BsaI sites.

For every calcium carbonate molecule precipitated, one calcium ion leaves the solution. Calcium carbonate is insoluble in water and hence, this calcium ion can no longer interact with other chemicals in the solution. If we know the initial calcium ion concentration, we can perform a Patton-Reeder calorimetric assay to find the resulting calcium concentration. The difference between the initial and final calcium concentrations equates the calcium that left the solution either to the cell or to be bound to calcium carbonate.

Figure 10: Calcium ion assay. TU1B was the same as TU1, except additional BsaI sites.

It is clear that the TU1 uptakes significantly more calcium ions from the environment, combined with the observed increase in the dry aggregate weight, this proves enhanced CaCO₃ precipitation.

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 BamHI site found at 1
  Illegal BamHI site found at 192
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 2813
  Illegal BsaI.rc site found at 2797