Part:BBa_K4417017

CuO-RBS-ureABC-rrnB T1 Terminator TU1B in pCT5c TUM

Description

The coding sequence of ureABC with additional BsaI sites (BBa_K4417015) was cloned into pCT5c (BBa_K4417000) to create the master plasmid BBa_K4417017. Besides, this composite plasmid contains a cumate inducible promoter (BBa_K4417007), strong RBS (BBa_K4417008), and an rrnB T1 terminator (BBa_K4417011).

Figure 1:Master plasmid construct.

Usage and Biology

• This composite part can be used to produce urease accessory proteins.
• This part is a master plasmid. It could be used to:
  • Create the urease nature operon construct (BBa_K4417018) with ureEFG TU2B (BBa_K4417016).
  • Create the multiple transcriptional urease units (BBa_K4417019) with TU1 (BBa_K4417012) and TU2 (BBa_K4417013).
• NdeI restriction can be used to change the promoter.

Cloning Strategy

BamHI and SacI sites were used to clone pCT5c plasmid with ureABC (BBa_K4417015) 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:Cloning strategy of the master plasmid- CuO-RBS-ureABC-rrnB T1 Terminator TU1B in pCT5c TUM.

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. TU1B was exactly the same as TU1, except two additional BsaI sites.

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. TU1B was exactly the same as TU1, except two additional BsaI sites.

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 (DH5a). 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
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 2658
  Illegal BsaI.rc site found at 2642