Difference between revisions of "Part:BBa K3376013"

Revision as of 07:10, 7 October 2020

BioBrick / pDL278

pDL278, a E. coli/Streptococcus shuttle vector

pDL278 is a shuttle vector between E. coli and Gram-positive bacteria esp. for Streptococcus spp., which was created by Donald J. LeBlanc, et al. in 1992. It could be applied for cloning vectors in E. coli and transforming S. mutans with a spectinomycin resistance cassette 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). We got the plasmid from the lab of Dr. Yuqing Li at Sichuan University in China. Firstly, we made the pDL278 compatible to BioBrick assembly system (i.e., EcoRI-XbaI-insert-SpeI-PstI). We amplified the backbone by PCR and did 2 rounds of site-directed mutagenesis with the primers listed in Fig. 1, C. The resulting plasmid was checked by restriction enzymes (Fig. 1, B) and further confirmed by sequencing.

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)

Electroporation in BTX™ Gemini X2 Electroporation System

↓ 40 µl of ice-cold electrocompetent cells in a 1-mm gap cuvette

↓ 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. 2 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)

Sequence and Features