Revision as of 13:38, 13 October 2022

CuO-RBS-ureABC-ureEFG-rrnB T1 Native Operon Sequence

Description

This part was created by cloning the master plasmid (BBa_K4417017) with the coding sequence of ureEFG (BBa_K4417016). This plasmid retained the nature urease operon sequence under a novel cumate inducible promoter (BBa_K4417007). The Type IIS backbone also contains a strong RBS (BBa_K4417008) and an rrnB T1 terminator (BBa_K4417011).

Figure 1:Urease nature operon construct from Sporosarcina pasteurii.

Usage and Biology

• This part can be transformed in both B. subtilis and E. coli.
• Inducer: p-isopropyl benzoate (cumate).
• Cumate is non-toxic to the host. In our experiment, we induced the urease expression with 50 μM cumate.
• E. coli ori is a pMB1 derivative
• B. sub ori is unknown
• The copy number of this plasmid in B. subtilis and E. coli is unknown.
• NdeI restriction can be used to change the promoter.

Cloning Strategy

Golden Gate Assembly was used to assemble the master plasmid with TU2B.

Figure 2:CuO-RBS-ureABC-ureEFG-rrnB T1 Terminator Nature Operon.

Method

We used Golden Gate Assembly to clone the ureEFG part into the master plasmid, following NEB’s Golden Gate Assembly Protocol (https://international.neb.com/protocols/2015/03/04/golden-gate-assembly-protocol-for-using-neb-golden-gate-assembly-mix-e1600) using BsaI restriction site.

Golden Gate Assembly protocol:

• The following reagents were assembled in a thin-walled PCR tube.
  • 0.5 μL T4 DNA Ligase
  • 2 μL of 10X T4 Ligase buffer
  • 0.5 μL BsaI enzyme
  • 100ng of receiver plasmid
  • Equimolar amounts of inserts
  • MilliQ for a total volume of 20 μL
• Mixed gently.
• Set up the thermocycler program.

• 5 μL of product was transformed into 50 μL competent cells. Beta-mercaptoethanol (bME) was used to improve the transformation efficiency following NEB’s protocol (https://international.neb.com/faqs/0001/01/01/how-can-i-increase-transformation-efficiency)

Figure 3:E. coli plates transformed with urease nature operon plasmid. (a), (b) transformed with no bME. (c), (d) transformed with 24mM bME.

Figure 3 verified that bME could be used to improve the transformation efficiency to a certain extent. After successful transformation, colonies were picked, grown, and the plasmid isolated. The isolated plasmid was checked by SapI diagnostic digest. 5 μL uncut and 10 μL cut samples were loaded on a 1% agarose gel to check the band size. From Figure 4, expected bands can be observed around 6880bp and 4789bp.

Figure 4:Restriction digest of urease nature operon plasmid. 1: DNA ladder, 2,4,6,8: nature operon plasmid cut with SapI, 3,5,7,9: nature operon plasmid uncut, 10: TU1 cut with SapI, 11: TU1 uncut.

Characterization

In order to observe whether the ureABCEFG 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, and ureF was identified at 24.9 kDa.

Sequence and Features