Revision as of 11:18, 12 October 2022

CuO-RBS-ureABC-rrnB T1 Terminator TU1

Description

The coding sequence of ureABC (BBa_K4417009) 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.

Figure 1: Composite part: CuO-RBS-ureABC-rrnB T1 Terminator TU1.

Usage and Biology

• This composite part can be used to produce urease structural complex ureABC.
• The part was synthesized by IDT as 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 TU1.

Construct TU1 was ligated with ureABC (BBa_K4417013) and pCT5c (BBa_K4417000). In Figure 2, the cloned plasmid was checked by diagnostic digest. Correct band size was observed of 6880bp and 2757bp.

Figure 3: Diagnostic digest of construct TU1. 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 TU1 was further verified from Sanger sequencing.

Figure 4: Part of the TU1 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 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.

Sequence and Features