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

2.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  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

GCTGGAATTCTCTAGAATTTCATGGTGATTCACGTTTA
AATTTAAAAAATG TACGTTAATAGAAATAATTCCTCCG

plnA promoter synthesis RP

CGTAACATCCCGGGCACCTCGCTTTTAGGATAATGT
GTTTTTGAAGTACGGAGGAATTATTTCTATTAACG

gusA  FP

GGTGCCCGGGATGTTACGTCCTGTAGAAACCC

gusA  RP

ATACGAATTCGCGGCCGCTTATTGTTTGCCTCCCTGC

 

 

 

Signal peptide-flag tag-DD13 RIP

F102001

CCCGGGAGGAGGCTCGAGATGGACTACAAAGACGAT
GACGATAAAG

R102002

TCGACTTTATCGTCATCGTCTTTGTAGTCCATCTCGAGC
CTCCTCCCGGGAGCT

F102003

TCGACATGGCTCTGTGGAAGACGCTGCTGAAGAAAGT
TCTGAAGGCTTATTCAC

R102004

CATGGTGAATAAGCCTTCAGAACTTTCTTCAGCAGCGT
CTTCCACAGAGCCATG

F102005

CATGGACGAATTTTTGAGCGGCCGCGGTAC

R102006

CGCGGCCGCTCAAAAATTCGTC

F102007

CCCGGGGATGGGAGCGACGTTAGATGAAAGAAGTAA
GGTTTTGGGGACTCTTGTTAGGAC

R102008

ACAAGAGTCCCCAAAACCTTACTTCTTTCATCTAACGTC
GCTCCCATCCCCGGGAGCT

F102009

TGTTTGTCTGTTTAGGGGCAGTGATACCGTTAGTTAGTA
AGGCTGACGTAG

R102010

TCGACTACGTCAGCCTTACTAACTAACGGTATCACTGCC
CCTAAACAGACAAACAGTCCTA

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. [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. [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. [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. [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.

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