Difference between revisions of "Part:BBa K3376013"

Latest revision as of 09:21, 13 October 2020

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.

The plasmid map in VectorNTI format (BioBrick compatible pDL278 vector - Mingdao iGEM2020.gb) can be DOWNLOAD here.

The sequence of the plasmid in the Part Registry has omitted the sequence of the insertion of BBa_J04450 with BioBrick Prefix and BioBrick Suffix.

Transformation of E. coli DH5alpha

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).

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

↓ 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)

Reference

1. LeBlanc DJ, Lee LN, Abu-Al-Jaibat A. Molecular, genetic, and functional analysis of the basic replicon of pVA380-1, a plasmid of oral streptococcal origin. Plasmid. 1992 Sep;28(2):130-45.

2. Gong T, Tang B, Zhou X, Zeng J, Lu M, Guo X, Peng X, Lei L, Gong B, Li Y. Genome editing in Streptococcus mutans through self-targeting CRISPR arrays. Mol Oral Microbiol. 2018 Dec;33(6):440-449.

3. Dunny GM, Lee LN, LeBlanc DJ. Improved electroporation and cloning vector system for gram-positive bacteria. Appl Environ Microbiol. 1991 Apr;57(4):1194-201.

4. Lima BP, Kho K, Nairn BL, Davies JR, Svensäter G, Chen R, Steffes A, Vreeman GW, Meredith TC, Herzberg MC. Streptococcus gordonii Type I Lipoteichoic Acid Contributes to Surface Protein Biogenesis. mSphere. 2019 Dec 4;4(6):e00814-19.

5. Correia FF, McKay TL, Farrow MF, Rosan B, DiRienzo JM. Natural transformation of Streptococcus crista. FEMS Microbiol Lett. 1996 Sep 15;143(1):13-8.

6. Gentry DR, Ingraham KA, Stanhope MJ, Rittenhouse S, Jarvest RL, O'Hanlon PJ, Brown JR, Holmes DJ. Variable sensitivity to bacterial methionyl-tRNA synthetase inhibitors reveals subpopulations of Streptococcus pneumoniae with two distinct methionyl-tRNA synthetase genes. Antimicrob Agents Chemother. 2003 Jun;47(6):1784-9.

7. Wu S, Liu Y, Lei L, Zhang H. Virulence of methicillin-resistant Staphylococcus aureus modulated by the YycFG two-component pathway in a rat model of osteomyelitis. J Orthop Surg Res. 2019 Dec 12;14(1):433.

8. Loimaranta V, Tenovuo J, Koivisto L, Karp M. Generation of bioluminescent Streptococcus mutans and its usage in rapid analysis of the efficacy of antimicrobial compounds. Antimicrob Agents Chemother. 1998 Aug;42(8):1906-10.

Sequence and Features