Team:Mingdao/Basic Part
Mingdao
BIOBRICK / pDL278
This year we created a basic part of the plasmid backbone with the features of shuttle vector between Gram(-) and Gram(+) bacteria. We performed several site-directed mutagenesis to generate a BioBrick-compatible plasmid backbone. We also demonstrated the feasibility of application in the SynBio field to satisfy the needs for engineering in broad host range.
pDL278, a E. coli/Gram(+) SHUTTLE VECTOR
pDL278 is a shuttle vector between E. coli and Gram-positive bacteria, which was created by Donald J. LeBlanc, et al. in 1992. It contains a spectinomycin resistance cassette for selection and the origin of replication of pBR322 for E. coli and ori (+) for Gram(+) bacteria, respectively. Ori (+) is from S. aureus with an ORF encoding an undefined protein possibly for plasmid replication (Fig. 1, A).
APPLICATION IN SYNTHETIC BIOLOGY
The pDL278 vector is used widely across Gram(-) and Gram(+) bacterial strains. In addition to E. coli, E. faecalis, another Gram(-) Enterococcus strain, was transformed with the vector by Gary M. Dunny, et al. For Gram(+) bacteria, several groups have demonstrated the transformation efficiency with pDL278 in Streptococcus gordonii by Bruno P. Lima, et al., in Streptococcus crista by Frederick F. Correia, et al., in Streptococcus pneumoniae by Daniel R. Gentry, et al., as well as in Staphylococcus aureus by Shizhou Wu, et al.
CONSTRUCTION FOR BIOBRICK-COMPATIBLE pDL278 VECTOR
We believe it's worth creating a BioBrick-compatible pDL278 vector for application as a transforming tool in the projects among iGEM community. We got the plasmid from the lab of Dr. Yuqing Li at Sichuan University in China. Firstly, we amplified the pDL278 plasmid by PCR and assembled with the standard part of BBa_J04450 (a RFP Coding Device) in pSB1C3 to create EcoRI-XbaI and SpeI-PstI cloning sites for further BioBrick assembly.
Unfortunately, a PstI site on the pDL278 disrupts the expression of the ORF for plasmid replication, and two XbaI sites are located in the promoter region for spectinomycin resistance gene expression. We conducted 2 rounds of site-directed mutagenesis by PCR with the primers listed in Fig. 1, C. to modify these restriction enzyme sites for BioBrick assembly. The resulting Biobrick-compatible plasmid was obtained and checked by restriction enzymes (Fig. 1, B) and further confirmed by sequencing.
Unfortunately, a PstI site on the pDL278 disrupts the expression of the ORF for plasmid replication, and two XbaI sites are located in the promoter region for spectinomycin resistance gene expression. We conducted 2 rounds of site-directed mutagenesis by PCR with the primers listed in Fig. 1, C. to modify these restriction enzyme sites for BioBrick assembly. The resulting Biobrick-compatible plasmid was obtained and checked by restriction enzymes (Fig. 1, B) and further confirmed by sequencing.
Fig. 1. BioBrick compatible pDL278 E. coli/Streptococcus shuttle vector. (A) Key features in pDL278 (B) Restriction enzyme check with a 1kb DNA ladder (C) Primers for site-directed mutagenesis and sequencing.
TRANSFORMATION OF E. coli DH5α
E. coli DH5alpha competent cells can be efficiently transformed using the traditional heat-shock method. The transformants carrying the J04450/pDL278 vector can be selected with 50 ug/ml of spectinomycin and express a red color in the colonies (Fig. 2). The mini-prep of plasmid concentration we usually got from overnight culture is around 200 ng/ul (260/280 = ~1.8, 260/230 > 4).
Fig. 2. Transformation of E. coli DH5alpha competent cells with the plasmid of J04450/pDL278.
TRANSFORMATION OF S. MUTANS BY ELECTROPORATION
We test transforming S. mutans by electroporation based on Vuokko Loimaranta’s protocol, briefly described as follows.
Prepare electrocompetent cells
↓ Cultivate S. mutans in BHI to OD600 of 0.6
↓ Wash twice with ice-cold buffer (10mM HEPES (pH 7.0), 15% glycerol)
↓ Resuspend in the electroporation buffer (5% sucrose, 15% glycerol)
↓ Wash twice with ice-cold buffer (10mM HEPES (pH 7.0), 15% glycerol)
↓ Resuspend in the electroporation buffer (5% sucrose, 15% glycerol)
Electroporation in BTX™ Gemini X2 Electroporation System
↓ 40 µl of ice-cold electrocompetent cells in a 1-mm gap cuvette
↓ 1 µl of pDL278-based plasmid DNA (~10ng)
↓ A single electric pulse of 4.5 ms (setting: 1.25 kV, 25µF, 200Ω)
↓ Immediately add fresh 960 ul of BHI broth
↓ After 1 hr, plate the cells onto BHI agar plate supplemented with 1 mg/ml of spectinomycin
↓ Grown for 2 days at 37°C, check the colony by PCR
↓ 1 µl of pDL278-based plasmid DNA (~10ng)
↓ A single electric pulse of 4.5 ms (setting: 1.25 kV, 25µF, 200Ω)
↓ Immediately add fresh 960 ul of BHI broth
↓ After 1 hr, plate the cells onto BHI agar plate supplemented with 1 mg/ml of spectinomycin
↓ Grown for 2 days at 37°C, check the colony by PCR
The gel data shown in Fig. 3 indicated the successful transformation of S. mutans by colony PCR with primer sets against the plasmid vector (pDL278-F/R) and gDNA of S. mutans (Mut-F/R)
