Team:HKUST/Project/Materials and Methods

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Team: HKUST

1.Vectors and bacterial strains
2.Building of chimeric constructs
3.GUS reporter assay
4.Protocol list

1.Vectors and bacterial strains

The bacterial strains and vectors used in iGEM HKUST 2010 are listed in Table 1 below. E. coli DH10B and DH5α cells are prepared as chemically competent cells and used for transformation. The E. coli cells are plated on antibiotic-containing agar plate and incubated at 37C overnight. L. plantarum WCFS1 used in our project are grown in MRS medium at 30C [1]. The antibiotic concentration we used are listed as follow: ampicillin (150μg/ml for E. coli), kanamycin (50μg/ml for E. coli), chloramphenicol (35μg/ml for E. coli), erythromycin (200μg/ml for E. coli, 10μg/ml for L. plantarum).

Table 1. Bacterial Strains and Vectors

Strains	Description	Source
E.coli DH10B	F- endA1 glnV44 thi-1 recA1 relA1 gyrA96 deoR nupG Φ80dlacZΔM15 Δ(lacZYA-argF)U169, hsdR17(rK- mK+), λ–; used for subcloning	HKUST stock
E.coli DH5α	F- endA1 recA1 galE15 galK16 nupG rpsL ΔlacX74 Φ80lacZΔM15 araD139 Δ(ara,leu)7697 mcrA Δ(mrr-hsdRMS-mcrBC) λ-; used for subcloning	HKUST stock
L. plantarum WCFS1	Wild type; used for device characterization	[2]
Non-virulent S. aureus	Used for device characterization	HKUST stock

Vectors	Description	Source
pBluescript KSII (+)	E. coli cloning vector containing T3 promoter, T7 promoter, lacZ gene with multiple cloning site inside; Ampr	HKUST stock
pBluescript SKII (+)	E. coli cloning vector containing T3 promoter, T7 promoter, lacZ gene with multiple cloning site inside. Multiple cloning site inverted; Ampr	HKUST stock
pMG36e	Shuttle vector contain P32 constitutive promoter; Emr	Yrbio Company
PBI121	Containing gusA reporter gene; Kmr	HKUST stock
PMH4	Containing mCherry fluorescent gene; Ampr	HKUST stock
pRN9232	Containing agrC-I; Ampr	[3]
pRN9233	Containing agrC-IV; Ampr	[3]
pRN6683	P3-blaZ fusion, for device characterization; Methr	[4]
BBa_I746101	Containing agrC-1; Kmr	iGEM 2010 BioBrick distribution
BBa_J04450	Containing RFP reporter gene; Ampr	iGEM 2010 BioBrick distribution

Building of chimeric constructs

1) Construction of chimeric sensor agrC-plnB in pBluescript and pMG36e

By PCR amplification of Biobrick BBa_I746101, the trans-membrane signal sensing domain agrC with 8 base pair-overhang of the cytoplasmic HPK domain of plnB was obtained. The cytoplasmic HPK domain of plnB with an overhang of agrC was also obtained by PCR amplification from extracted L. plantarum WCFS1 genomic DNA. Next, fusion PCR of two gene fragments was conducted and the desired fusion gene agrC-plnB obtained. After SacI and KpnI double digestion, aforementioned agrC-plnB fusion gene was ligated into pBluescript KSII (+). The cloning vector was transformed into E. coli DH10B. In subsequent steps, the construct agrC-plnB was taken out from pBluescript KSII (+) by SacI and KpnI double digestion, and ligated into E. coli – Lactobacillus shuttle vector pMG36e. The parts were confirmed by DNA sequencing.

2) Construction of agrC-mCherry and agrC-plnB-mCherry in pBluescript and pMG36e

After SacI and KpnI double digestion, agrC-mCherry and agrC-plnB-mCherry fusion gene were ligated into pBluescript KSII (+). The cloning vector was transformed into E. coli DH10B. In subsequent steps, the constructs agrC-mCherry and agrC-plnB-mCherry were taken out from pBluescript KSII (+) by SacI and KpnI double digestion, and ligated into E. coli – Lactobacillus shuttle vector pMG36e. The parts were confirmed by DNA sequencing.

3)Construction of plnA promoter – gusA reporter unit in pBluescript

The 124-base-pair nucleotide of plnA promoter was obtained by annealing a 79-base-pair forward primer and a 73-base-pair reverse primer. The reporter gene gusA was amplified from PBI121 by PCR. The gusA gene was then digested and ligated into pBluescript KSII (+). The cloning vector was transformed into E. coli DH10B. However, we tried several times but still failed to ligate the 124 bp plnA promoter into pBluescript KSII (+).

4)Construction of “hybrid signal peptide-flag tag-DD13-RIP”

Signal peptide, flag-tag and DD13-RIP were obtained by annealing single-stranded oligonucleotides. Each fragment was flanked by restriction enzyme cutting sites for subsequent ligation. Construct “signal peptide- flag tag- DD13 –RIP” was ligated into pBluescript KSII (+). The cloning vector was transformed into E. coli DH10B plasmid amplification. The parts were confirmed by DNA sequencing.

Table 2. Primer List
Construct	Name	Sequence
agrC	agrC FP	CAGCTAGAGCTCAAAGAGGAGAAATACTAGATGATTCTG
agrC	agrC RP	GACTCGGTACCTCTTTGGATCCTTATTAGTTATTGATG

agrC-plnB	agrC FP	CAGCTAGAGCTCAAAGAGGAGAAATACTAGATGATTCTG
	agrC RP1	GTTTCTAACAGGAACTGGCTGATAACGAAAG
	plnB FP	CAGTTCCTGTTAGAAACGATTAGAGTATATGCTTGGC
	plnB RP1	GTCAGGTACCTTATTTATCCTCCGTAACAATTAACG

agrC-mCherry	agrC FP	CAGCTAGAGCTCAAAGAGGAGAAATACTAGATGATTCTG
	agrC RP 2	ATTGCGGCGTTATTGATGATTTCGACTTTCTGAATG
	mCherry FP 2	ATCAATAACGCCGCAATGGTGAGCAAG
	mCherry RP	ATCGTCCCGGGGCTTACTTGTACAGCTCGTCCATG

agrC-plnB-mCherry	agrC FP	CAGCTAGAGCTCAAAGAGGAGAAATACTAGATGATTCTG
	agrC RP 1	GTTTCTAACAGGAACTGGCTGATAACGAAAG
	plnB FP	CAGTTCCTGTTAGAAACGATTAGAGTATATGCTTGGC
	plnB RP	ATTGCGGCTTTATCCTCCGTAACAATTAACGTC
	mCherry FP 1	GAGGATAAAGCCGCAATGGTGAGCAAG
	mCherry RP	ATCGTCCCGGGGCTTACTTGTACAGCTCGTCCATG

plnA promoter-gusA	plnA promoter synthesis FP	GCTGGAATTCTCTAGAATTTCATGGTGATTCACGTTTAAATTTAAAAAATG TACGTTAATAGAAATAATTCCTCCG
	plnA promoter synthesis RP	CGTAACATCCCGGGCACCTCGCTTTTAGGATAATGTGTTTTTGAAGTACGGAGGAATTATTTCTATTAACG
	gusA FP	GGTGCCCGGGATGTTACGTCCTGTAGAAACCC
	gusA RP	ATACGAATTCGCGGCCGCTTATTGTTTGCCTCCCTGC

Signal peptide-flag tag-DD13 RIP	F102001	CCCGGGAGGAGGCTCGAGATGGACTACAAAGACGATGACGATAAAG
	R102002	TCGACTTTATCGTCATCGTCTTTGTAGTCCATCTCGAGCCTCCTCCCGGGAGCT
	F102003	TCGACATGGCTCTGTGGAAGACGCTGCTGAAGAAAGTTCTGAAGGCTTATTCAC
	R102004	CATGGTGAATAAGCCTTCAGAACTTTCTTCAGCAGCGTCTTCCACAGAGCCATG
	F102005	CATGGACGAATTTTTGAGCGGCCGCGGTAC
	R102006	CGCGGCCGCTCAAAAATTCGTC
	F102007	CCCGGGGATGGGAGCGACGTTAGATGAAAGAAGTAAGGTTTTGGGGACTCTTGTTAGGAC
	R102008	ACAAGAGTCCCCAAAACCTTACTTCTTTCATCTAACGTCGCTCCCATCCCCGGGAGCT
	F102009	TGTTTGTCTGTTTAGGGGCAGTGATACCGTTAGTTAGTAAGGCTGACGTAG
	R102010	TCGACTACGTCAGCCTTACTAACTAACGGTATCACTGCCCCTAAACAGACAAACAGTCCTA

3.GUS reporter assay

pMG36e-transformed L. plantarum WCFS1 is cultivated at 30C until its optical density at 600nm (OD600) reaches 0.10-0.20. Cells will then be exposed to autoinducing peptide (AIP) and incubated at 30C for 1-2 hours. GUS substrate para-nitrophenyl-β-D-glucuronic acid will then be added and the reaction mixture incubated at 37C for another 20 minutes. Finally, OD reading at 405nm obtained and GUS activity quantified as OD405 / OD600 [1].

Protocol list

Heat-shock competent cell preparation heat-shock transformation
http://www.promega.com/guides/subcloning_guide/_row/transforming_bacteria_row.pdf
Electro-competent cell preparation and electro-transformation
http://www.its.caltech.edu/~bjorker/Protocols/Prep_of_electocomp_cells.pdf
Mini-prep
FavorPrep™ Plasmid DNA Extraction Mini Kit
Midi-prep
FavorPrep™ Plasmid DNA Extraction Midi/Maxi Kit
Genomic DNA extraction from L.plantarum
http://www.springerlink.com/content/j2g06724067924p4/#section=86476&page=1
PCR
http://openwetware.org/wiki/PCR
Fusion PCR
http://www.fgsc.net/Aspergillus/Oakley_PCR_protocol.pdf
Colony PCR
http://openwetware.org/wiki/Endy:Colony_PCR
Restriction digest
http://openwetware.org/wiki/Silver:_Restriction_Digest
Ethanol precipitation
http://openwetware.org/wiki/Ethanol_precipitation_of_nucleic_acids
Kit purification
FavorPrep™ GEL/PCR Purification Mini Kit
Gel purification
FavorPrep™ GEL/PCR Purification Mini Kit
Vector dephosphorylation
http://www.neb.com/nebecomm/products_intl/protocol76.asp
Ligation
http://openwetware.org/wiki/DNA_Ligation
Agarose gel electrophoresis
http://www.methodbook.net/dna/agarogel.html
DNA PAGE (Polyacrylamide gel electrophoresis)
http://microbiology.ucdavis.edu/heyer/protocols/dna%20page.pdf
GUS assay
[1]
Flag-tag assay and western blot
http://www.biomol.de/details/AD/protocol-GPCR-WB.pdf

References:

[1] Johnsborg, O., Diep, D. B. & Nes, N. F. (2003). Structural analysis of the peptide pheromone receptor plnB, a histidine protein kinase from Lactobacillus plantarum. Journal of Bacteriology, 185 (23), 6913–6920.
[2] Kleerebezem, M., Boekhorst, J., Kranenburg, R.V., Molenaar, D. & Kuipers, O.P. (2003) Complete genome sequence of Lactobacillus plantarum WCFS1. Proceedings of the National Academy of Sciences, 100(4), 1990-1995.
[3] Geisinger, E., George, E.A., Muir, T.W, & Novick, R.P. (2008) Identification of ligand specificity determinants in agrC, the Staphylococcus aureus quorum-sensing receptor. 283(14), 8930–8938.
[4] Novick, R.P., Projan, S.J., Kornblu, J., Ross, H.F. & Ji, G. (1995) The agr P2 operon: an autocatalytic sensory transduction system in Staphylococcus aureus. 248(4), 446-458.