Team:Heidelberg/Notebook/material

From 2008.igem.org

(Difference between revisions)

Latest revision as of 09:15, 20 August 2009

Material

Buffers & Solution


SM	50 mM	Tris, pH 7.5
	20 mM	MgSO4
	50 mM	NaCl
	0.01 %	gelatine

Tethering buffer, pH 7.0	10 mM	potassium phosphate
		(K2HPO4 : KH2PO4 = 1 :1 )
	100 µM	EDTA
	1 µM	L-Methionine
	10 mM	lactic acid

M63 salt	3 g/l	KH2PO4
	9 g/l	K2HPO4
	4 g/l	(NH4)2SO4
	0.5 g/l	FeSO4

Amino acid mix	5 g/l	L-threonine
	5 g/l	L-histidin
	5 g/l	L-leucine
	5 g/l	L-methionine

M9 salts	64 g/l	Na2HPO4 x 7H2O
	15 g/l	KH2PO4
	2.5 g/l	NaCl
	5 g/l	NH4Cl

[back]

Kits

Kit	supplier

CompactPrep Plasmid Maxi Kit	Qiagen
HISpeed Plasmid Maxi Kit	Qiagen
MaxPlax™ Lambda Packaging Extracts	EPICENTRE Biotechnologies
QIAEX II Gel Extraction Kit	Qiagen
QIAGEN Lambda Mini Kit	Qiagen
QIAprep Spin Mini Kit	Qiagen
QIAquick Gel Extraction Kit	Qiagen
QIAquick PCR Purification Kit	Qiagen

[back]

Marker

Marker	supplier

GeneRuler™ High Range DNA Ladder	MBI Fermentas
GeneRuler™ 1kb DNA Ladder Mix	MBI Fermentas
GeneRuler™ 1kb Plus DNA Ladder Mix	MBI Fermentas

[back]

Enzymes

Enzym	supplier

Pfu DNA polymerase	Stratagene
Pfu turbo DNA polymerase	Stratagene
Phusion DNA polymerase	Finnzymes
Taq DNA polymerase	MBI Fermentas
T4 DNA ligase	MBI Fermentas / New England Biolabs

AgeI	New England Biolabs
BamHI	New England Biolabs
BglI	MBI Fermentas
BseJI	MBI Fermentas
BspEI	New England Biolabs
DpnI	Roche Diagnostics / New England Biolabs
DraI	New England Biolabs
EcoRI	New England Biolabs
EcoRV	New England Biolabs
HindIII	New England Biolabs
KpnI	New England Biolabs
NcoI	New England Biolabs
NdeI	New England Biolabs
NotI	New England Biolabs
PstI	New England Biolabs
SacI	New England Biolabs
SalI	MBI Fermentas
ScaI	New England Biolabs
SfcI	New England Biolabs
SmiI	MBI Fermentas
SpeI	New England Biolabs
SpeI	New England Biolabs
XbaI	New England Biolabs
XhoI	MBI Fermentas
XmaI	New England Biolabs

[back]

Plasmidvectors

Name	application	reference

[http://partsregistry.org/wiki/index.php?title=Part:BBa_B0015 BBa_B0015]	terminator	http://partsregistry.org
[http://partsregistry.org/wiki/index.php?title=Part:BBa_F1610 BBa_F1610]	LuxI	http://partsregistry.org
[http://partsregistry.org/wiki/index.php?title=Part:BBa_I15030 BBa_I15030]	AI-1 amplifier	http://partsregistry.org
[http://partsregistry.org/wiki/index.php?title=Part:BBa_I20260 BBa_I20260]	GFP	http://partsregistry.org
[http://partsregistry.org/wiki/index.php?title=Part:BBa_J01003 BBa_J01003]	oriT	http://partsregistry.org
[http://partsregistry.org/wiki/index.php?title=Part:BBa_J16002 BBa_J16002]	cloning vector	http://partsregistry.org
[http://partsregistry.org/wiki/index.php?title=Part:BBa_J23107 BBa_J23107]	constitutive promotor	http://partsregistry.org
[http://partsregistry.org/wiki/index.php?title=Part:BBa_T9002 BBa_T9002]	AI-1 GFP receiver	http://partsregistry.org
CheY-mCherry	cloning vector	V. Sourjik, ZMBH
ColE1	colicin E1	DSMZ
ColE9-J	colicin E9	C. Kleanthous, University of York
pBad18	cloning vector	V. Sourjik, ZMBH
pBad33	cloning vector	V. Sourjik, ZMBH
pBluescript II SK (+)	cloning vector	V. Sourjik, ZMBH
pDest15	cloning vector	DKFZ Library
pDK4	visualization	V. Sourjik, ZMBH
pDK48	cloning vector	V. Sourjik, ZMBH
pDK58	cloning vector	V. Sourjik, ZMBH
pDK6	visualization	V. Sourjik, ZMBH
pED374	oriT	K. Derbyshire, Wadsworth
pES16	cloning vector	V. Sourjik, ZMBH
pMMB863	oriT	M. Bagdasarian, MSU
pQE-30	cloning vector	Invitrogen
pSB1A2	cloning vector	http://partsregistry.org
pSB2K3	cloning vector	http://partsregistry.org
pTrc99a	cloning vector	V. Sourjik, ZMBH
pUB307	helper plasmid	E. Lanka, BfR
RP4	helper plasmid	M. Bagdasarian, MSU

[back]

Synthetic oligonucleotides

All oligonucleotides were purchased from Invitrogen (Karlsruhe) and adjusted to a standard concentration of 100 ^pmol/_µl.

Name	SEQUENCE (5´->3´)

Bam_fw	GACAAGTGTTGGCCATGGAACAGG
Bam_rv	GCCGTCTGTGATGGCTTCCATG
cI_mut_fw	GCGTCTGGGTGGTGATGAGTTCACCTTCAAAAAACTG
cI_mut_rv	CAGTTTTTTGAAGGTGAACTCATCACCACCCAGACGC
CmR_EcoRI_mut_fw	GAATGCTCATCCGGAGTTCCGTATGGCAATG
CmR_EcoRI_mut_rev	CATTGCCATACGGAACTCCGGATGAGCATTC
CmR_fw	GCTAAAATGGAGAAAAAAATCACTGG
CmR_new_fw	TACGAGGTACCTTTACAGCTAGCTCAGTCCTAGGTATTATGC
CmR_new_rev	TATATAAGCTTTTACGCCCCGCCCTGCCACTCATCGCAGTACTGTTG
CmR_Prefix_fw	GAATTCGCGGCCGCTTCTAGAGTTTACAGCTAGCTCAGTCCTAGG
CmR_rv	AGGTTCTCCTTTATTAGCCGGATCCTCTAGATTACGCC
CmR_Suffix_rv	CTGCAGCGGCCGCTACTAGTATATAAACGCAGAAAGGCCCACCC
colE1_kil_prot_rv_A_SpeI	TATATACTAGTACTACTGAACCGCGATCCCCG
colE1_mut_EcoRI_fw	GGTATTGCTATTGTTACAGGTATTCTATGCTCCTATATTGATAAG
colE1_mut_EcoRI_rv	CTTATCAATATAGGAGCATAGAATACCTGTAACAATAGCAATACC
colE1_mut_PstI_1_fw	GCAGTAAAAGTGAAAGTTCAGCAGCTATTCATGCAACTGC
colE1_mut_PstI_1_rv	GCAGTTGCATGAATAGCTGCTGAACTTTCACTTTTACTGC
colE1_mut_PstI_2_fw	GCTGCCCGGGCAAAAGCAGCAGCGGAAGCACAGG
colE1_mut_PstI_2_rv	CCTGTGCTTCCGCTGCTGCTTTTGCCCGGGCAGC
colE1_mut_PstI_3_fw	CATTAGAGAAGAAAGCAGCAGATGCAGGGGTGAG
colE1_mut_PstI_3_rv	CTCACCCCTGCATCTGCTGCTTTCTTCTCTAATG
colE1_prot_fw_BamH1	TACGAGGATCCATGGAAACCGCGGTAGCG
colE1_prot_rv_HindIII	TATATAAGCTTTTAAATCCCTAACACCTC
colE9_lysProt_rv_A_SpeI	TATATACTAGTACTAGGTTTTCGGCTTAGAACCCC
colE9_mut_EcoRI_fw	GTTGGGTGGACGATTCGAGAGTTCAATGGGGAAATAAAAATG
colE9_mut_EcoRI_rv	CATTTTTATTTCCCCATTGAACTCTCGAATCGTCCACCCAAC
colE9_plasmid_rv_A_SpeI	TATATACTAGTACACATGGAACTTTTGTGAC
colE9_prot_fw_BamH1	TACGAGGATCCATCGATTTGCCCATGACCC
colE9_prot_rv_XmaI	TATATCCCGGGTTACTTACCTCGGTGAATATCG
DK13	TCACCCGCACGCGC
DK167	AGGATACTAGTAGGCCATTACTTT
DK9b	CGATGCGGCCGCTCAAAATGTTTCCCAGTTTGG
F1610_fw_XbaI	TACGATCTAGAAAAGAGGAGAAATACTAG
F1610_rv_HindIII	TATATAAGCTTTATAAACGCAGAAAGGCCC
GAM_fw	AGTGCTTTAGCGTTAACTTCCG
GAM_rv	GGTTTTACCGCATACCAATAACG
GFP_CmR_fw	CTCGTTGGTACCTCTAGATTTACAGCTAGCTCAGTCCTAGG
GFP_CmR_rv	TATTCGACCGGTACTAGTTATAAACGCAGAAAGGCCCACC
GFP_new_fw	TACGAGAGCTCTTTACAGCTAGCTCAGTCCTAGG
GFP_new_rv	TATATACCGGTACTAGTTATAAACGCAGAAAGGCCCACC
Lambda_insert_fw	TTGTAAAAACAGCCCTCCTC
Lambda_insert_rv	GATATGACTATCAAGGCCGC
LuxP_mut_F	CGTGAATTAGCAACAGAGTTCGGAAAGTTCTTCCC
LuxP_mut_R	GGGAAGAACTTTCCGAACTCTGTTGCTAATTCACG
LuxP_prefix_F	GAGGGAGAATTCGCGGCCGCTTCTAGATGAAGAAAGCGTTACTATTTTC
LuxP_sufffix_R	GGAGAGCTGCAGCGGCCGCTACTAGTAATTATCTGAATATCTAAATGCG
LuxPc	ATTACGCGGCCGCAGGAAACAGACCATGAAGAAAGCGTTACTATTTTCCC
LuxPd	GTAATGTCGACTCAATTATCTGAATATC
LuxP-seq-FW	CCCGTCCTGCCAGTGAGC
LuxQa	ATCGACCATGGGCAATAAATTTCGCTTAGC
LuxQc	GTAATGGATCCTTAGTGGAGGCTTGAGCC
LuxQTar_1a	CACAAATCATTGCCAATGAACGTATGTTGCTTACTCCGCTGG
LuxQTar_1b	CCAGCGGAGTAAGCAACATACGTTCATTGGCAATGATTTGTG
LuxQTar_2a	CTTAGCGACCATGAGCCATGAGTTTGCCCAGTGGCAACTGGC
LuxQTar_2b	GCCAGTTGCCACTGGGCAAACTCATGGCTCATGGTCGCTAAG
LuxS mutBbaIR	CCACACCCACTTCTAGGATGTTCTTCGCGATTTGC
LuxS mutXbaIF	GCAAATCGCGAAGAACATCCTAGAAGTGGGTGTGG
LuxS_mut_fw	CATCCTTTCTGAGAAAGGCATTCATACATTAGAGC
LuxS_mut_rev	GCTCTAATGTATGAATGCCTTTCTCAGAAAGGATG
LuxS_prefix_F	GGAGAGGAATTCGCGGCCGCTTCTAGATGGGCAATGCACCAGCGGTTCG
luxS_suffix_R	GAGGGACTGCAGCGGCCGCTACTAGTAGTCGATGCGTAGCTCTCTCAGC
LuxSa	ATCAGTCCATGGGCAATGCACCAGCGGTTCG
LuxSb	GTAATGGATCCTTAGTCGATGCGTAGC
mut_insert_fw	CTGAGGGGACGGTACCTCTACATTTACAGCTAGCTCAG
mut_insert_rv	CTGAGCTAGCTGTAAATGTAGAGGTACCGTCCCCTCAG
mut_insert2_fw	GGCAGGCGGGGCGTAATCTATAGGATCCGGCTAATAAAGG
mut_insert2_rv	CCTTTATTAGCCGGATCCTATAGATTACGCCCCGCCTGCC
mut_kpn1_pBlue	CGAGGGGGGGCCCGGTTCCCAATTCGCCCTATAG
mut_kpn1_pBlue	CTATAGGGCGAATTGGGAACCGGGCCCCCCCTCG
oriT_pre	GAATTCGCGGCCGCTTCTAGAGGACAGGCTCATGCCGGCCGC
oriT_RP4_fw	CTCGTTTCTAGAACTAGTGACAGGCTCATGCCGGCCGC
oriT_RP4_rv	TATTCGGGTACCGTCCCCTCAGTTCAGTAATTTCCTGC
oriT_suf	CTGCAGCGGCCGCTACTAGTAGTCCCCTCAGTTCAGTAATTTCCTGC
T9002_Lux_pR_rv_SpeI_BamHI_RBS	TATATACTAGTGGATCCGGTTCTGTTTCCTCTCTAGTATTTATTCGAC
T9002_LuxpR_Not_Eco_Xba_G_fw	TACGAGAATTCGCGGCCGCTTCTAGAGTCCCTATCAGTGATAGAGATTG
Term_new_fw	TACGAAAGCTTCCAGGCATCAAATAAAACGAAAGG
Term_new_rv	TATATGAGCTCTATAAACGCAGAAAGGCCCACCC
VF2	TGCCACCTGACGTCTAAGAA
VIC121	TTTATCGCAACTCTCTACTG
VIC122	CTGATTTAATCTGTATCAGG
VIC131	ATGTGTGGAATTGTGAGCGG
VIC132	CTGATTTAATCTGTATCAGG
VR	ATTACCGCCTTTGAGTGAGC

[back]

Phages

Name	application	reference

Lambda cI mut	cI deleted	W. Reiser, ZMBH
Lambda cI857	heat inducible	MBI Fermentas

[back]

Bacteria

E.coli strain	usage	reference

DH5a	amplification of plasmids	Invitrogen
HCB33	swarm assays	V. Sourjik, ZMBH
MG1655	swarm assays	V. Sourjik, ZMBH
TOP10	amplification of plasmids	Invitrogen
UU1250	chemotaxis receptor knock out	V. Sourjik, ZMBH

[back]

Bacteria Growth Media

Luria Broth (LB)	10 ^g/_l	tryptone
	5 ^g/_l	yeast extract
	10 ^g/_l	NaCl

Luria Broth (LB) plus	10 ^g/_l	tryptone
	5 ^g/_l	yeast extract
	20 ^g/_l	NaCl

TB	10 ^g/_l	bacto tryptone
	5 ^g/_l	NaCl

Standard I	15.6 ^g/_l	peptone P
	2.8 ^g/_l	yeast extract
	5.6 ^g/_l	NaCl
	1 ^g/_l	D-glucose

Minimal medium	9.8 %	M63 salts
	0.2 %	glycerol
	0.1 ^g/_l	Thiamine
	1 mM	MgSO₄
	0.8 %	amino acid mix

M9	20 %	M9 salts
	2 mM	MgSO₄
	0.4 %	glucose
	0.1 mM	CaCl₂

For cultivation of phages the media were supplied with 0.2 % maltose and 10 mM MgSO4. For preparation of agar plates 15 g/l agar, for preparation of top agar 7 g/l agar was added prior the autoclaving. For selection of resistant bacteria following antibiotics were added during cooling of at agar at about 50 °C:

[back]

Antibiotic	concentration

Ampicillin	100 µg/ml
Chloramphenicol	35 µg/ml
Kanamycin	50 µg/ml

The same concentrations of antibiotics were used for selection of resistant in media.

[back]

Methods

Preparing chemically competent cells

First, a 20 ml over night culture was inoculated in antibiotic free LB medium from a fresh single colony and transferred into 400 ml antibiotic free LB medium the next day. This culture was incubated at 37 °C while shacking until an OD600 of 0.5 – 0.6 was achieved. The culture was than cooled down on ice, centrifuged (8 min, 4 °C, 3500 rpm), the supernatant discarded and the pellet resuspended in 10 ml 100 mM CaCl₂. After addition of further 190 ml 100 mM CaCl₂ the suspension was incubated on ice for 30 min. The suspension was than again centrifuged (8 min, 4 °C, 3500 rpm), the supernatant discarded, the pellet resuspended in 20 ml 82.5 mM CaCl2 with 17.5 % glycerol and aliquoted. The aliquots were flash frozen in liquid nitrogen and than stored at -80 °C until usage. [back]

Transformation of bacteria

For enrichment of vectors, E .coli TOP10 and DH5α were used. For the transformation 100 µl of the competent cells were thawed on ice and 50 – 200 ng DNA solution added (depending on the concentration of the DNA solution). After a 20 minute incubation on ice, cells were made permeable for the DNA by heat shocking for 45 seconds at 42 °C and a further 2 minute incubation on ice. The samples were than rescued by adding 250µl preheated antibiotic free LB-medium and incubated for one hour at 37 °C while shacking for induction of the antibiotic resistance. The selection for plasmid containing and therefore antibiotic resistant bacteria was conducted by plating them on antibiotic containing LB-agar plates. [back]

Isolation of plasmid DNA by alkaline lysis (mini and maxiprep)

For analysis of ligations and transformations QIAprep Spin Kits (Qiagen, Hilden) were used following the manufacturer instructions. For miniprep a single colony was picked from a LB-agar plate or glycerol stock was used to inoculate 5 ml LB-medium with appropriate antibiotic for selection (100 µg/µl ampicillin, 50 µg/µl kanamycin, 35 µg/µl chloramphenicol). Bacteria were grown over night at 37 °C while shaking (200 rpm). By using 4 ml over night culture with this kit the yield was around 6-10 µg. For maxipreps the Qiagen CompactPrep Plasmid Maxi Kit was used following the instructions given by the instruction manual. In this case 250 ml LB-medium were inoculated and used for preparation of plasmid DNA. The routinely yield was 300-400 µg plasmid DNA. Purity and amount of DNA was analysed using a NanoDrop. [back]

DNA amplification using polymerase chain reaction (PCR)

By using PCR smallest amount of DNA can be detected and amplified. The principle of PCR is the selective amplification of any region of the DNA. Nevertheless, the sequence at both ends must be known for the binding of two complementary primers. The DNA region of interest is than amplified exponentially while the reproduction of the DNA takes place in three temperature stpes: denaturation of parental DNA at high temperature (95-98 °C), hybridisation of primers (58-65 °C) and DNA elongation (72 °C). For the amplification a heat-stable DNA polymerase I with a 3’-5’ exonuclease activity (proof reading) is used such as Pfu (from Pyrococcus furiosus) and Phusion (a Pyrocuccus-like enzyme) or the non proof reading enzyme Taq (from Thermus aquaticus). Phsuion polymerase was used in a 2x master mix adding only primers (20pmol each) and DNA template (~10ng) or alternatively colonies from agar plates (1-5 colonies) in a final volume of 50 µl. For taq and Pfu polymerase following reaction batch was commonly used:


1 µl template (10 ng) / colonies
2 µl primer 1 (10 pmol/µl)
2 µl primer 2 (10 pmol/µl)
1 µl polymerase (2,5 Units)
1 µl dNTP mix (10 mM)
5 µl 10x buffer
38 µl dH2O

The PCR procedure was as follows:


Initiale denaturation	95- 98 °C, 3-5 min	1 cycle

denaturation	95-98 °C, 15 sec - 1 min
annealing	58-65 °C, 15 sec - 1 min	25-28 cycles
elongation	72 °C, 15 sec - 3 min

termination	72 °C, 5 – 10 min	1 cycle
	4 °C	forever

For site directed mutagenesis PCR Pfu turbo polymerase (Stratagene) was used in the same reaction batch described above. The temperature program was as follows:


initiale denaturation	95 °C, 30 sec	1 cycle

denaturation	95 °C, 30 sec
annealing	55 °C, 1 min	16 cycles
elongation	68 °C, 2 min per kb of plasmid

termination	68 °C, 10 min	1 cycle
	4 °C	forever

For point mutagenesis primers with around 33 bases were used having the base to be altered in the middle. The melting temperature of these primers was always over 78 °C. If this melting temperature could not be achieved using 15 unchanged nucleotides at both sites the flanking arms were enlarged properly. After the PCR reaction 10 units of DpnI was added directly to the PCR tube, mixed and incubated for 1-5 h at 37 °C. DpnI digests the parental methylated plasmid DNA leaving only the mutated one. After purification using QIAquick PCR Purification Kit the plasmid was transformed as described above. [back]

Purification of DNA from PCR reactions

PCR products were purified by the QIAquick PCR Purification Kit from Qiagen following the instructions of the Qiagen Handbook. To check the purity and amount of extracted DNA an aliquot was analysed using a NanoDrop. [back]

Enzymatic hydrolysis of DNA by restriction enzymes

The restriction digest of DNA was used for analysis of purified DNA form mini or maxiprep or for isolation of specific DNA fragments for further cloning. Analytical digestions were routinely conducted in 20 µl volume. In all digestions a minimum of 2 Units restriction enzyme(s) was used per microgram DNA. Optimised buffer conditions were secured by using NEB buffer system. The final reaction volume was achieved by adding H₂O dest. and the sample was incubated at optimal temperature for the restriction enzyme(s) (normally 37 °C). Preparative digestions were conducted in a volume of 50 µl. For analysis and preparation DNA loading dye was added to the samples and they were loaded on a agarose gel or alternatively purified by QIAquick PCR Purification Kit from Qiagen. [back]

Agarose gel electrophoresis for separating DNA

In the agarose gel electrophoresis a mixture of DNA fragments with different sizes are separated in an electrical field by their size. This is achieved by moving the negatively charged DNA through an agarose matrix while shorter fragments will run faster. The size of the pores can be controlled by agarose concentration. The higher the agarose concentration the smaller the pores are and the smaller fragments can be separated. Agarose concentrations between 0.7 and 1.5 % agarose in 0.5x TE buffer were used. The agarose was dissolved completely by heating up and 0.1 µg/ml ethidium bromide was added. The DNA fragments were separated using a constant voltage between 80 and 130 V. Under UV light (λ = 254 nm) DNA is visible through the unspecific intercalated ethidium bromide and can be documented or cut out and extracted from the gel. [back]

Isolation of DNA fragments from an agarose gel

Plasmid DNA and DNA fragments were extracted using the Gel Extraction Kit from Qiagen following the manufacture instructions. To check the purity and amount of extracted DNA an aliquot was analysed using a NanoDrop. [back]

Ligation of dsDNA fragments

T4 DNA ligase was used to form covalent phosphodiesterbonds between doublestranded DNA fragments having blunt or compatible sticky ends. For the ligation the restricted vector -DNA and the insert were mixed 1:3 or 1:5 in a total ligationvolume of 20 µl. This mixture was incubated in ligation buffer using 5 Weiss units T4 DNA ligase over night at 16 °C or at room temperature for 20 minutes and afterwards used to transform chemical competent cells. [back]

Lysing of bacteria by ultrasonication

For the lysis of bacteria an ultrasonic tip was used. The 10-15 ml of bacteria over night cultures were sonicated with an ultrasonic tip three time for 15 seconds. After sonication cell extract was observed under microscope. [back]

Glycerol stock

To store bacteria for long term glycerol stocks were used. Therefore 1 ml of an over night culture were added to 150 µl of 80 % Glycerol into a cryo tube, vortexed and incubated at room temperature for 30 min. Afterwards the glycerole stock was stored at -80 °C. [back]

Preparation of plating bacteria for bacteriophage experiments

A overnight culture of the appropriate E. coli strain was grown in LB medium containing 10 mM MgSO₄ and 0.2 % maltose at 30 °C to reduce the amount of cell debris in the medium. The added maltose leads to a substantial induction of the maltose operon including the lamb gene, which encodes the cell surface receptor to which bacteriophage λ binds. After harvesting the cells they were resuspended in 10 mM MgSO₄ and diluted to a final concentration of 2.0 OD₆₀₀. The suspension of plating bacteria was stored at 4 °C for up to 1 week. [back]

Bacteriophage λ plaque assay

Tenfold serial dilutions of the bacteriophage stocks were prepared, 100 µl of it mixed with the same amount of plating bacteria, incubated for 20 minutes at 37 °C to allow the bacteriophage particles to adsorb to the bacteria, this mixture added to 3 ml molten top agar which was kept liquid at 48 °C and the entire contend poured onto a agar plate. After harden of the top agar the inverted plates were incubated at 37 °C over night. On the next day plaques could be counted. [back]

Preparing Stocks of Bacteriophage λ

Completely lysed agarose plates were prepared by mixing 0.1 ml plating bacteria with ~10⁵ pfu, incubation for 20 minutes at 37 °C and that bringing out the mixture in 3 ml molten top agarose. Agarose is preferred in this protocol in order to minimize contaminations with polyanionic inhibitors that can interfere with subsequent enzymatic analysis of the bacteriophage DNA. The plates were incubated 12-16 hours without inversion at 37 °C. Plates were not inverted during incubation to encourage sweating of fluid onto the surface of the dish, which allows the bacteriophage to spread more easily. The plates were than overlayed with 5 ml SM and incubated while shaking at 4 °C over night. The SM was that transferred to a tube and the plate washed again with 1 ml SM. 2 % v/v chloroform was added and bacterial debris removed by centrifugation at 10,000 g for 10 minutes at 4 °C. The concentration of infectious particles in the stock was measured by plaque assays as described above. [back]

Preparing of λ DNA

Lambda DNA was prepared using the QIAGEN® Lambda mini kit following the manufacture instructions. [back]

In vitro packaging of λ DNA

In vitro packaging was conducted using the MaxPlax lambda packaging extracts from Epicentre Biotechnologies following the manufacture instructions. [back]

Colicin Activity Test

To measure the killing efficiency and which amount of cells or colicins are needed to reach any killing activity a colicin activity test was carried out. Therefore bacteria containing the colicin plasmid (E. coli TOP10 or E. coli MG1655) and GFP producing cells (reference promoter [http://partsregistry.org/wiki/index.php?title=Part:BBa_I20260 BBa_I20260], E. coli TOP10) were inoculated in TB-media with appropriated antibiotics at 37 °C for 4 to 6 hours and the optical densities of the two strains were adapted. The colicin cells themselves, their produced supernatant or the supernatant of the lysed colicin cells were added in different ratios to a constant amount of GFP producing cells. The total volume was kept constant and the missing amount added with TB-media without antibiotics. The colicin production was induced by several concentrations (0 M-100 nM) of N-Acyl-Homoserin-Lactone (AHL). The OD and GFP intensities were measured at 37 °C in the Tecan Microplate Reader every half an hour for about 12 hours. As a negative control the similar test was carried out with cells, containing the same plasmid without the colicin operon on it. [back]

Lysis test

Lysis of killercells dependent on the AHL concentration was measured due to a lysis test. Therefore E. coli TOP 10 containing the luxR-colicinE1 part ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K150009 BBa_K150009]) and a GFP producing part ([http://partsregistry.org/wiki/index.php?title=Part:BBa_I20260 BBa_I20260]) were inoculated in TB-media with appropriate antibiotics at 37 °C for 4 to 6 hours. As reference E. coli TOP 10 cells containing a luxR part (comparable to [http://partsregistry.org/wiki/index.php?title=Part:BBa_F2620 BBa_F2620]) instead of the luxR-colicinE1 part ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K150009 BBa_K150009]). For the lysis measurement 250 µl of the liquid cultures were diluted with 250 µl TB-media. The Lux pR promoter was activated with AHL concentrations from 0 M up to 50 nM. The optical densities and GFP intensities were measured in Tecan Microplate Reader every half-hourly for about 12 hours. [back]

Sender Activity Test

The sender activity test was performed to quantify the AHL production of the cells containing the sender part ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K150000 BBa_K150000]). Therefore TB-media with the appropriated antibiotic was inoculated with 2 µl/ml of an overnight culture. For 7 hours every hour the OD of the medium was measured and 5 ml of the culture were spin down to produce supernatant. The sterile filtered supernatant, still containing the AHL, was stored at 4 °C. The supernatant of every hour was then added in different ratios to a constant amount of AHL inducible cells which produce GFP after induction. The OD and GFP intensities were measured at 37 °C in the Tecan Microplate Reader every half an hour for about 12 - 24 hours. The same test was carried out with amplifier cells ([http://partsregistry.org/wiki/index.php?title=Part:BBa_I15030 BBa_I15030]). To calculate the produced amount of AHL by sender or amplifier cells as reference the test were performed also with different AHL concentration (0 M-100 nM) instead of AHL producing cells. [back]

LuxS Activity Test

This protocol for the functional check AI-2 production is mainly adapted from a paper of Vanderleyden et al. [1] , for further questions please refer to it. 10ml ONC DH5α strain with LuxS (S) and 10ml DH5α strain with ptrc99α as blank (B) were incubated in LB to a volume of 50mL each at 37°C up to an OD of 0.35 in 100ml erlenmeyer flasks. Then S and B each were induced with 500 µM IPTG for 3h, following centrifugation at maximum speed for 20min. The supernatants were then transferred in two new falcons by pipetting or decanting. Optional the supernatant was sterile filtrated and/or 10 g/L NaCl was added to a final concentration of ~20g/L. 10 mL ONC of V. harveyi reporter (BB120 in LB plus at 30 °C) was incubated in LB plus to a final volume of 50 mL at 37°C up to an OD of 0.35 in a 100 mL erlenmeyer flask. Supernatant S and B were both mixed with Vibrio cells at ratios 1:3, 1:5 and 1:10 each to a final volume of 5 mL in 50 mL falcon tubes and incubated for 30min at 30°C. Subsequently the cells were incubated again for 3h, OD and Luminescence were measured with a microplate reader. [back]

[1] Sigrid C. J. DeKeersmaecker and Jos Vanderleyden ‘Constraints on detection of autoinducer-2 (AI-2) signalling molecules using Vibrio harveyi as a reporter’, Microbiology 2003 149: 1953-1956.

Conjugation test

Equal amounts of donor (cells harbouring the oriT containing plasmid and pUB307) and acceptor cells from overnight cultures were centrifuged (2 min, 13000 rpm), the pellet washed twice with 1.5 ml LB medium without antibiotics, the pellets combined and this mixture resuspended in 100 µl LB medium without antibiotics. This cell mix was transferred to a filter membrane on a LB agar plate and incubated here for a time range from 0 to 42 minutes in 6 minutes steps at 37 °C. The membrane was then transferred in 1 ml LB medium, vortexed, the medium diluted and plated out on LB agar plates with proper antibiotics for plasmid harbouring the oriT and antibiotic resistance of acceptor bacteria. On the next day the number of transconjugants was counted and therewith the conjugation efficiency calculated as ratio of cell density of transconjugants to cell density of donor cells depending on the reaction time. [back]

Receptor Polarization Test

To check the functionality of the fusion receptor constructs each construct, on pDK48, was transformed into strain UU1250 together with pDK36 (YFP-CheA). The expression of the receptor was induced with 0.01% arabinose, of YFP-CheA with 30µM IPTG. Polarization was tested by putting 10 µL of the cells on a 0.5% agarose pad and making snapshots using a Zeiss Axio Imager.Z1 fluorescent microscope.

Swarm Assays

The sensitivity of the LuxP-tar fusion constructs to AI-2 was tested in swarm assays. For this two different strains were used: E. coli UU1250 and E. coli HCB33. The assays were performed in TB medium for UU1250 and TB or minimal medium for HCB33 (0.3% agar). As chemo attractant either 3 µl of an AI-2 producing ONC, induced with 100 nM IPTG, or 10 µl supernatant from those cells was spotted 13 times on the plate to create a center line. Gradient building was allowed for 12-16 hours at 4 °C. Afterwards the fusion receptor expressing cells (0.01% arabinose induced overnight) as well as non-chemotactic reference strains were spotted at a distance of about 3-3.5 cm to the attractant line. Swarm plates were incubated at 30 °C and pictures taken after 24, 48 and 72 hours. [back]

Microscopy – Chemotaxis

An Applied Precision - DeltaVision wide field microscope was used to visualize chemotactic behaviour of the LuxP-tar fusion. E. coli UU1250 and E. coli HCB33 were used for these measurements. The monitoring was performed with Labtek^TM chambered coverglass systems (Nunc) which were filled with 2 ml of TB media, Tethering Buffer and minimal media containing 0.2 % agar. As chemo attractant either 3 µl of an AI-2 producing ONC, induced with 100 nM IPTG, or 10 µl supernatant from those cells was spotted at the one side of the chamber. Gradient building was allowed for 12-16 hours at 4 °C. Afterwards either fusion receptor expressing cells (0.01% arabinose induced overnight) or non-chemotactic reference strains were spotted at the center of the chamber. The chamber was then incubated for several hours at 30 °C. After 3 hours frame pictures were taken, using confocal fluorescence imaging and 8x6 frames. (8 pictures in length, 6 in height) Images were processed and auto arranged with housemade ImageJ plugins. Matlab was used for the quantitative analysis. [back]

Microscopy – Colicin Toxicity

The killing efficiency of colicin producing cells was monitored using an Applied Precision - DeltaVision wide field microscope. Therefore a prey and a killer strain were designed in E. coli TOP10. The prey strain contained a GFP producing part ([http://partsregistry.org/wiki/index.php?title=Part:BBa_I20260 BBa_I20260]) as well as the luxI sender part ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K150000 BBa_K150000]) and the killer strain contained as marker a mCherry producing part as well as the LuxR-colicinE1-receiver ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K150009 BBa_K150009]). Measurements were prepared with 1 ml of a prey ONC and 1 ml of a killer ONC in 3 ml TB media containing 0,01% arabinose and 10 nM AHL for induction of colicin production. A second probe in which cells without colicin genes were used instead of the killer cells was prepared in parallel. Both samples were incubated for 6-7 hours at 37 °C. Monitoring was performed on the Deltavision microscope with 8-well Labtek^TM chambered coverglass systems (Nunc). For that 6 µl of each sample were distributed equaly in 200 µl TB media. Frame pictures have been taken using 8x8 frames evenly distributed over the whole chamber. Images were processed and auto arranged with housemade ImageJ plugins. [back]

Microscopy – Cell lysis

To visualize the lysis of prey and killer cells cultures were observed on Zeiss Axio Imager.Z1 microscope. The killer strain (E. coli TOP 10) contained the luxR-colicinE1 part ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K150009 BBa_K150009]) and a mCherry producing part and the prey strain contained the senderpart ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K150000 BBa_K150000]) and a GFP producing part ([http://partsregistry.org/wiki/index.php?title=Part:BBa_I20260 BBa_I20260]). Both strains were inoculated in TB-media with appropriate antibiotics at 37 °C for 4 to 6 hours. Thereafter OD was adjusted and the killer strain was induced by 25 nM AHL. After 2 hour 200 µl of the induced killer culture and 200 µl of the prey culture were combined and centrifuged down. For microscopy pelleted cells were diluted with 10 µl and plated on a 0.5% agarosepad. Fixed positions were observed half hourly in the mCherry and GFP channel with a 100x oil immersion objective. [back]

Cell Culture

MCF-7 cells (breast cancer cell line) wt or stably expressing cytoplasmic GFP, were plated in 24 well plates (150 thousand cells per well) with 10 % FCS (fetal calf serum) DMEM (Dulbecco's Modified Eagle Medium) and incubated overnight at 37 °C, 5 % CO2. E. coli TOP 10 strains containing LuxR-colicin E1-receiver (BBa_K150009) and strains containing LuxR-receiver (no colicin) were grown overnight in TB media with appropriate antibiotics at 37 °C to an OD of 1.0 and diluted in the morning to 0.1. Once bacterial cells reached an OD of 0.35 they were harvested and inoculated on the MCF-7 wt and on MCF-7-GFP cells. [back]

Eucaryotic Killing Assay with Colicins

Once MCF-7 cells were confluent (12h after passage), E. coli TOP 10 bacteria were spinned down (1 ml of OD = 0.35) and resuspended with DMEM medium from the original wells containing the MCF-7 wt/MCF-7-GFP cells. A 24-well plate was used containing MCF-7 cells stained with Propidium Iodide (PI) alone, with LuxR-receiver cells not containing colicin and LuxR-receiver-colicin E1 cells. The second 24-well plate with MCF-7GFP was processed as described for wt MCF-7, but without any PI staining. AHL was added to every well to a final concentration of 5 nM. Well content was collected at different time-points (0 – 6 h) and when necessary, PI was added to the suspension 15 minutes before Flow Cytometer analysis. [back]

Flow Cytometer Acquisition

At the referred timepoints, supernatants were collected and spinned down. Eukaryotic cells were detached by trypsinization (200 μl) for 10 minutes at 37 °C and then resuspended in 800 μl of KH + 1 % BSA (Krebs Henseleit medium with 1 % Bovine Serum Albumin). After resuspending, well content was added to the precipitated supernatant to account for floating dead cells. PI was added to each sample (1:1000) and incubated at RT for 15 minutes before acquisition. PI positive cells and GFP expressing cells (%) were measured for 3000 cells for each condition. Histograms and fluorescent measurements were obtained using a Cytomics FC 500 MPL from Beckman&Coulter. [back]

@@ Line 1: / Line 1: @@
 <html>
-<link rel='stylesheet' href='http://igem-heidelberg.de/fileadmin/Wiki/Heidelberg2.css' type='text/css' />
-<link rel="stylesheet" href="http://igem-heidelberg.de/fileadmin/Wiki/Menu.css" type="text/css"/>
-<link rel="stylesheet"  href="http://igem-heidelberg.de/fileadmin/Wiki/MenuHD.css" type="text/css"/>
+<style>
+h1.firstHeading { display: none; }
+p {text-align: justify;}
+a:link { color: #00b0e6; text-decoration: none}
+a:visited { color:#00b0e6; text-decoration: none}
+a:hover { color:#f29400; text-decoration: none}
+a:active { color:#f29400; text-decoration: none}
+#bodyContent { padding: 10px auto; width: 910px; margin: auto; clear: none; }
+table#team_members { text-align: justify; border: 0; }
+table#team_members h2, table#team_members h3 { clear: both; }
+/*-----------------------------------------------------------------------------------------------*/
+div.MenuBar ul li ul.DropDownMenu {
+  display: none; /* Hides all drop-down menus. */
+}
+/*
+li:hover works in IE7 and FF2.
+a:hover works in IE6 and FF2.
+a:hover breaks li:hover in FF2.
+*/
+div.MenuBar ul li:hover ul.DropDownMenu li ul.SideMenu,
+div.MenuBar ul li a:hover ul.DropDownMenu li a ul.SideMenu {
+  display: none; /* Hides all side menus. */
+}
+/*------------------------------------------------------------------------------------- Menu Bar */
+div.MenuBar {
+  font: arial, helvetica, sans-serif;
+  height: 30px;
+  width: 910px;
+  /*width: 100%*/
+  margin: 0;
+  border-top: 0;
+  border-right: 0;
+  border-left: 0;
+  padding: 0;
+  background: black;
+}
+div.MenuBar ul {
+  font: arial, helvetica, sans-serif;
+  text-align: center;
+  list-style-type: none;
+  margin: 0.5em auto;
+  border: 0;
+  padding: 0;
+  background: black;
+}
+div.MenuBar ul li {
+  font: arial, helvetica, sans-serif;
+  display: block;
+  padding: 0;
+  margin: 0;
+  font-size: 1.3em;
+  float: left;
+  background: black;
+  text-align: center;
+  width: 107px;
+  position: relative; /* Sets the positioning context for each drop-down menu. */
+}
+div.MenuBar ul li a {
+  font: arial, helvetica, sans-serif;
+  display: block;
+  background: black;
+  height: 22px; /* Keep height + padding-top + padding-bottom sync with the menu bar height. */
+  color: #ffffff;
+  padding-top: 4px;
+  padding-bottom: 4px;
+  padding-left: 1em; /* Sets the left space between top-level items. */
+  padding-right: 1em; /* Sets the right space between top-level items. */
+  text-decoration: none;
+}
+/*------------------------------------------------------------------------------ Drop-Down Menus */
+div.MenuBar ul li:hover ul.DropDownMenu,
+div.MenuBar ul li a:hover ul.DropDownMenu {
+  display: block;
+  width: 10em; /* Drop-down menu width.
+                  Use MenuTailor.css to customize. */
+  height: 1em;
+  padding: 1px; /* Sets the drop-down menu "effective border" width. */
+  position: absolute;
+  top: 23px; /* Places the drop-down menu under the menu bar.
+                Keep it sync with the menu bar height. */
+  left: 0; /* Aligns the drop-down menu to its top-level item. */
+  background-color: black; /* Selected item. */
+  color: #FFFFFF;
+}
+div.MenuBar ul li:hover ul.DropDownMenu li a,
+div.MenuBar ul li a:hover ul.DropDownMenu li a {
+  width: 10em; /* Keep it sync with the drop-down menu width.
+                             Use MenuTailor.css to customize. */
+  height: 1em;
+  padding-left: 0;
+  padding-right: 0;
+  background-color: black; /* Selected item. */
+  color: #FFFFFF;
+}
+ul.DropDownMenu li a span {
+  display: block;
+  padding-left: 0.75em; /* Sets the left space of each drop-down menu item. */
+  padding-right: 0.25em; /* Sets the right space of each drop-down menu item. */
+  text-align: right; /* Aligns the >> symbol to the right. */
+}
+ul.DropDownMenu li a span span {
+  float: left; /* Aligns the text (back) to the left. */
+  font: 12px arial, helvetica, sans-serif; /* Required for IE55. */
+  height: 20px;
+  color: #FFFFFF;
+}
+/*----------------------------------------------------------------------------------- Side Menus */
+div.MenuBar ul li:hover ul.DropDownMenu li:hover ul.SideMenu,
+div.MenuBar ul li a:hover ul.DropDownMenu li a:hover ul.SideMenu {
+  display: block;
+  width: 11em; /* Side menu width.
+                  Use MenuTailor.css to customize. */
+  padding: 1px; /* Sets the side menu "effective border" width. */
+  position: absolute;
+  top: -1px; /* Aligns the side menu to its drop-down menu item.
+                Keep it sync with the side menu "effective border" width. */
+  left: 13em; /* Places the side menu to the right of the drop-down menu.
+                            Keep it sync with the drop-down menu width.
+                            Use MenuTailor.css to customize. */
+}
+div.MenuBar ul li:hover ul.DropDownMenu li:hover ul.SideMenu li a,
+div.MenuBar ul li a:hover ul.DropDownMenu li a:hover ul.SideMenu li a {
+  width: 11em; /* Keep it sync with the side menu width.
+                             Use MenuTailor.css to customize. */
+  font: 12px arial, helvetica, sans-serif; /* Required for IE55. */
+  left: 13em; /* Places the side menu to the right of the drop-down menu.
+                            Keep it sync with the drop-down menu width.
+                            Use MenuTailor.css to customize. */
+}
+div.MenuBar ul li ul.DropDownMenu li ul.SideMenu li a span {
+  padding-left: 1.5em; /* Sets the left space of each side menu item. */
+  padding-right: 0.5em; /* Sets the right space of each side menu item. */
+  text-align: left;
+  font: 12px arial, helvetica, sans-serif; /* Required for IE55. */
+  left: 13em; /* Places the side menu to the right of the drop-down menu.
+                            Keep it sync with the drop-down menu width.
+                            Use MenuTailor.css to customize. */
+}
+/*----------------------------------------------------------------------------- Browser Specific */
+* html div.MenuBar ul li a {
+  float: left; /* Required for IE55 and IE6.
+                             Breaks O9.
+                             Hidden (* html) from non-IE browsers. */
+}
+* html ul.DropDownMenu li a:hover {
+  cursor: hand; /* Required for IE55.
+                   Hidden (* html) from non-IE browsers. */
+}
+ul.DropDownMenu li a:hover {
+  cursor: pointer; /* Required for IE6 and IE7.
+                      Hidding it (* html) from non-IE browsers breaks IE7!
+}
+* html div.MenuBar a:hover {
+  text-decoration: none; /* Required for IE55 and IE6.
+                            Hidden (* html) from non-IE browsers. */
+}
+* html div.MenuBar ul li table,
+* html div.MenuBar ul li table td {
+  border: 0; /* Required for IE55 and IE6.
+                Hidden (* html) from non-IE browsers. */
+}
+/*------------------------------------------------------------------------------- Default Colors */
+div.MenuBar {
+  background-color: Menu;
+  border-bottom: 1px solid ButtonShadow;
+}
+div.MenuBar a {
+  background-color: Menu; /* Top-level unselected items. */
+  color: MenuText;
+}
+div.MenuBar ul li:hover a,
+div.MenuBar ul li a:hover {
+  color: #ea7f16;
+  background-color: Highlight; /* Top-level selected item. */
+  color: HighlightText;
+}
+/*...............................................................................................*/
+div.MenuBar ul li:hover ul.DropDownMenu,
+div.MenuBar ul li a:hover ul.DropDownMenu {
+  background-color: ButtonShadow; /* Sets the drop-down menu "effective border" color. */
+}
+div.MenuBar ul li:hover ul.DropDownMenu li a,
+div.MenuBar ul li a:hover ul.DropDownMenu li a {
+  background-color: Menu;  Drop-down menu unselected items.
+                             Sets the drop-down menu "effective background" color. */
+  color: MenuText;
+}
+div.MenuBar ul li:hover ul.DropDownMenu li:hover a,
+div.MenuBar ul li a:hover ul.DropDownMenu li a:hover {
+  background-color: Highlight; /* Drop-down menu selected item. */
+  color: HighlightText;
+}
+/*...............................................................................................*/
+div.MenuBar ul li:hover ul.DropDownMenu li:hover ul.SideMenu,
+div.MenuBar ul li a:hover ul.DropDownMenu li a:hover ul.SideMenu {
+  background-color: ButtonShadow; /* Sets the side menu "effective border" color. */
+}
+div.MenuBar ul li:hover ul.DropDownMenu li:hover ul.SideMenu li a,
+div.MenuBar ul li a:hover ul.DropDownMenu li a:hover ul.SideMenu li a {
+  background-color: Menu; /* Side menu unselected items.
+                             Sets the side menu "effective background" color. */
+  color: MenuText;
+}
+div.MenuBar ul li:hover ul.DropDownMenu li:hover ul.SideMenu li a:hover,
+div.MenuBar ul li a:hover ul.DropDownMenu li a:hover ul.SideMenu li a:hover {
+  background-color: Highlight; /* Side menu selected item. */
+  color: HighlightText;
+}
+/*-----------------------------------------------------------------------------------------------*/
+/*Script-Free 3-Level Menu 1.2 Tailor
+  www.CesarDaniel.info
+/*-------------------------------------------------------------------------------------- General */
+body {
+  background: white;
+  color: black;
+  margin: 0;
+  border: 0;
+  padding: 0;
+}
+div.MenuBar {
+  font: 13px arial, helvetica, sans-serif;
+}
+div.MenuBar ul {
+  font: 13px arial, helvetica, sans-serif; /* Required for IE55. */
+}
+/*--------------------------------------------------------------------------------------- Colors */
+div.MenuBar {
+  background-color: black; /* Selected item. */
+  color: #FFFFFF;
+  border-bottom: 1px solid ButtonShadow;
+}
+div.MenuBar a,
+div.MenuBar ul li:hover ul.DropDownMenu li a,
+div.MenuBar ul li a:hover ul.DropDownMenu li a,
+div.MenuBar ul li:hover ul.DropDownMenu li:hover ul.SideMenu li a,
+div.MenuBar ul li a:hover ul.DropDownMenu li a:hover ul.SideMenu li a {
+  background-color: black; /* Selected item. */
+  color: #FFFFFF;
+}
+div.MenuBar ul li:hover a,
+div.MenuBar ul li a:hover,
+div.MenuBar ul li:hover ul.DropDownMenu li:hover a,
+div.MenuBar ul li a:hover ul.DropDownMenu li a:hover,
+div.MenuBar ul li:hover ul.DropDownMenu li:hover ul.SideMenu li a:hover,
+div.MenuBar ul li a:hover ul.DropDownMenu li a:hover ul.SideMenu li a:hover {
+  background-color: #00b0e6; /* Selected item. */
+  color: #FFFFFF;
+}
+div.MenuBar ul li:hover ul.DropDownMenu,
+div.MenuBar ul li a:hover ul.DropDownMenu,
+div.MenuBar ul li:hover ul.DropDownMenu li:hover ul.SideMenu,
+div.MenuBar ul li a:hover ul.DropDownMenu li a:hover ul.SideMenu {
+  background-color: ButtonShadow; /* Sets the drop-down and side menus "effective border" color. */
+}
+/*--------------------------------------------------------------------------------------- Widths */
+/*
+/*
+Menu Bar 1
+Drop-Down Menu #2
+*/
+div.MenuBar#navi ul li:hover ul.DropDownMenu#MB1-DDM4,
+div.MenuBar#navi ul li a:hover ul.DropDownMenu#MB1-DDM4,
+div.MenuBar#navi ul li:hover ul.DropDownMenu#MB1-DDM4 li a,
+div.MenuBar#navi ul li a:hover ul.DropDownMenu#MB1-DDM4 li a {
+  width: 11em; /* Drop-down menu width. */
+}
+div.MenuBar#navi ul li:hover ul.DropDownMenu#MB1-DDM5,
+div.MenuBar#navi ul li a:hover ul.DropDownMenu#MB1-DDM5,
+div.MenuBar#navi ul li:hover ul.DropDownMenu#MB1-DDM5 li a,
+div.MenuBar#navi ul li a:hover ul.DropDownMenu#MB1-DDM5 li a {
+  width: 12em; /* Drop-down menu width. */
+}
+/*...............................................................................................*/
+/*
+Menu Bar 1
+Drop-Down Menu #2
+Side Menu #1
+*/
+div.MenuBar#navi ul li:hover ul.DropDownMenu li:hover ul.SideMenu#MB1-DDM2-SM1,
+div.MenuBar#navi ul li a:hover ul.DropDownMenu li a:hover ul.SideMenu#MB1-DDM2-SM1 {
+  left: 15.5em !important; /* Places the side menu to the right of the drop-down menu.
+                            Keep it sync with the drop-down menu width. */
+}
+div.MenuBar#navi ul li:hover ul.DropDownMenu li:hover ul.SideMenu#MB1-DDM2-SM1,
+div.MenuBar#navi ul li a:hover ul.DropDownMenu li a:hover ul.SideMenu#MB1-DDM2-SM1,
+div.MenuBar#navi ul li:hover ul.DropDownMenu li:hover ul.SideMenu#MB1-DDM2-SM1 li a,
+div.MenuBar#navi ul li a:hover ul.DropDownMenu li a:hover ul.SideMenu#MB1-DDM2-SM1 li a {
+  width: 10em; /* Side menu width. */
+}
+/*...............................................................................................*/
+/*
+Menu Bar 1
+Drop-Down Menu #2
+Side Menu #2
+*/
+div.MenuBar#navi ul li:hover ul.DropDownMenu li:hover ul.SideMenu#MB1-DDM2-SM2,
+div.MenuBar#navi ul li a:hover ul.DropDownMenu li a:hover ul.SideMenu#MB1-DDM2-SM2 {
+  left: 15.5em  !important; /* Places the side menu to the right of the drop-down menu.
+                            Keep it sync with the drop-down menu width. */
+}
+div.MenuBar#navi ul li:hover ul.DropDownMenu li:hover ul.SideMenu#MB1-DDM2-SM2,
+div.MenuBar#navi ul li a:hover ul.DropDownMenu li a:hover ul.SideMenu#MB1-DDM2-SM2,
+div.MenuBar#navi ul li:hover ul.DropDownMenu li:hover ul.SideMenu#MB1-DDM2-SM2 li a,
+div.MenuBar#navi ul li a:hover ul.DropDownMenu li a:hover ul.SideMenu#MB1-DDM2-SM2 li a {
+  width: 10em; /* Side menu width. */
+}
+/*...............................................................................................*/
+/*
+Menu Bar 1
+Drop-Down Menu #2
+Side Menu #3
+*/
+div.MenuBar#navi ul li:hover ul.DropDownMenu li:hover ul.SideMenu#MB1-DDM2-SM3,
+div.MenuBar#navi ul li a:hover ul.DropDownMenu li a:hover ul.SideMenu#MB1-DDM2-SM3 {
+  left: 15.5em  !important; /* Places the side menu to the right of the drop-down menu.
+                            Keep it sync with the drop-down menu width. */
+}
+div.MenuBar#navi ul li:hover ul.DropDownMenu li:hover ul.SideMenu#MB1-DDM2-SM3,
+div.MenuBar#navi ul li a:hover ul.DropDownMenu li a:hover ul.SideMenu#MB1-DDM2-SM3,
+div.MenuBar#navi ul li:hover ul.DropDownMenu li:hover ul.SideMenu#MB1-DDM2-SM3 li a,
+div.MenuBar#navi ul li a:hover ul.DropDownMenu li a:hover ul.SideMenu#MB1-DDM2-SM3 li a {
+  width: 10em; /* Side menu width. */
+}
+/*...............................................................................................*/
+</style>
 <body>
@@ Line 122: / Line 463: @@
 </html>
-==Materials & Methods==
-In progress..
+==Material==
+===Buffers & Solution===
+{| class="wikitable"
+|- bgcolor=grey
+! height=20px, width=200px |  || width=250px |  || width=250px |
+|-align="center"
+| style="font-weight:bold;" |SM || 50 mM || Tris, pH 7.5
+|-align="center"
+| style="font-weight:bold;" | || 20 mM || MgSO4
+|-align="center"
+| style="font-weight:bold;" | || 50 mM || NaCl
+|-align="center"
+| style="font-weight:bold;" | || 0.01 % || gelatine
+|-style="height:20px"
+|  ||  ||
+|-align="center"
+| style="font-weight:bold;" |Tethering buffer, pH 7.0 || 10 mM  || potassium phosphate
+|-align="center"
+| style="font-weight:bold;" |  ||  || (K2HPO4 : KH2PO4 = 1 :1 )
+|-align="center"
+| style="font-weight:bold;" | || 100 µM || EDTA
+|-align="center"
+| style="font-weight:bold;" | || 1 µM || L-Methionine
+|-align="center"
+| style="font-weight:bold;" | || 10 mM || lactic acid
+|-style="height:20px"
+|  ||  ||
+|-align="center"
+| style="font-weight:bold;" |M63 salt || 3 g/l || KH2PO4
+|-align="center"
+| style="font-weight:bold;" | || 9 g/l || K2HPO4
+|-align="center"
+| style="font-weight:bold;" | || 4 g/l || (NH4)2SO4
+|-align="center"
+| style="font-weight:bold;" | || 0.5 g/l || FeSO4
+|-style="height:20px"
+|  ||  ||
+|-align="center"
+| style="font-weight:bold;" |Amino acid mix || 5 g/l || L-threonine
+|-align="center"
+| style="font-weight:bold;" | || 5 g/l || L-histidin
+|-align="center"
+| style="font-weight:bold;" | || 5 g/l || L-leucine
+|-align="center"
+| style="font-weight:bold;" | || 5 g/l || L-methionine
+|-style="height:20px"
+|  ||  ||
+|-align="center"
+| style="font-weight:bold;" |M9 salts || 64 g/l || Na2HPO4 x 7H2O
+|-align="center"
+| style="font-weight:bold;" | || 15 g/l || KH2PO4
+|-align="center"
+| style="font-weight:bold;" | || 2.5 g/l || NaCl
+|-align="center"
+| style="font-weight:bold;" | || 5 g/l || NH4Cl
+|-
+|}
+[[https://2008.igem.org/Team:Heidelberg/Notebook/material back]]
+===Kits===
+{| class="wikitable"
+|- bgcolor=grey
+! height=20px, width=350px | Kit || width=350px | supplier
+|-style="height:20px"
+|  ||
+|-align="center"
+| style="font-weight:bold;" |CompactPrep Plasmid Maxi Kit || Qiagen
+|-align="center"
+| style="font-weight:bold;" |HISpeed Plasmid Maxi Kit || Qiagen
+|-align="center"
+| style="font-weight:bold;" |MaxPlax™ Lambda Packaging Extracts || EPICENTRE Biotechnologies
+|-align="center"
+| style="font-weight:bold;" |QIAEX II Gel Extraction Kit || Qiagen
+|-align="center"
+| style="font-weight:bold;" |QIAGEN Lambda Mini Kit || Qiagen
+|-align="center"
+| style="font-weight:bold;" |QIAprep Spin Mini Kit  || Qiagen
+|-align="center"
+| style="font-weight:bold;" |QIAquick Gel Extraction Kit || Qiagen
+|-align="center"
+| style="font-weight:bold;" |QIAquick PCR Purification Kit || Qiagen
+|-
+|}
+[[https://2008.igem.org/Team:Heidelberg/Notebook/material back]]
+===Marker===
+{| class="wikitable"
+|- bgcolor=grey
+! height=20px, width=350px | Marker || width=350px | supplier
+|-style="height:20px"
+|  ||
+|-align="center"
+| style="font-weight:bold;" |GeneRuler™ High Range DNA Ladder || MBI Fermentas
+|-align="center"
+| style="font-weight:bold;" |GeneRuler™ 1kb DNA Ladder Mix || MBI Fermentas
+|-align="center"
+| style="font-weight:bold;" |GeneRuler™ 1kb Plus DNA Ladder Mix || MBI Fermentas
+|-
+|}
+[[https://2008.igem.org/Team:Heidelberg/Notebook/material back]]
+===Enzymes===
+{| class="wikitable"
+|- bgcolor=grey
+! height=20px, width=350px | Enzym || width=350px | supplier
+|-style="height:20px"
+|  ||
+|-align="center"
+| style="font-weight:bold;" |Pfu DNA polymerase || Stratagene
+|-align="center"
+| style="font-weight:bold;" |Pfu turbo DNA polymerase || Stratagene
+|-align="center"
+| style="font-weight:bold;" |Phusion DNA polymerase || Finnzymes
+|-align="center"
+| style="font-weight:bold;" |Taq DNA polymerase || MBI Fermentas
+|-align="center"
+| style="font-weight:bold;" |T4 DNA ligase || MBI Fermentas / New England Biolabs
+|-style="height:20px"
+|  ||
+|-align="center"
+| style="font-weight:bold;" |AgeI || New England Biolabs
+|-align="center"
+| style="font-weight:bold;" |BamHI || New England Biolabs
+|-align="center"
+| style="font-weight:bold;" |BglI || MBI Fermentas
+|-align="center"
+| style="font-weight:bold;" |BseJI || MBI Fermentas
+|-align="center"
+| style="font-weight:bold;" |BspEI || New England Biolabs
+|-align="center"
+| style="font-weight:bold;" |DpnI || Roche Diagnostics / New England Biolabs
+|-align="center"
+| style="font-weight:bold;" |DraI || New England Biolabs
+|-align="center"
+| style="font-weight:bold;" |EcoRI || New England Biolabs
+|-align="center"
+| style="font-weight:bold;" |EcoRV || New England Biolabs
+|-align="center"
+| style="font-weight:bold;" |HindIII || New England Biolabs
+|-align="center"
+| style="font-weight:bold;" |KpnI || New England Biolabs
+|-align="center"
+| style="font-weight:bold;" |NcoI || New England Biolabs
+|-align="center"
+| style="font-weight:bold;" |NdeI || New England Biolabs
+|-align="center"
+| style="font-weight:bold;" |NotI || New England Biolabs
+|-align="center"
+| style="font-weight:bold;" |PstI || New England Biolabs
+|-align="center"
+| style="font-weight:bold;" |SacI || New England Biolabs
+|-align="center"
+| style="font-weight:bold;" |SalI || MBI Fermentas
+|-align="center"
+| style="font-weight:bold;" |ScaI || New England Biolabs
+|-align="center"
+| style="font-weight:bold;" |SfcI || New England Biolabs
+|-align="center"
+| style="font-weight:bold;" |SmiI || MBI Fermentas
+|-align="center"
+| style="font-weight:bold;" |SpeI || New England Biolabs
+|-align="center"
+| style="font-weight:bold;" |SpeI || New England Biolabs
+|-align="center"
+| style="font-weight:bold;" |XbaI || New England Biolabs
+|-align="center"
+| style="font-weight:bold;" |XhoI || MBI Fermentas
+|-align="center"
+| style="font-weight:bold;" |XmaI || New England Biolabs
+|-
+|}
+[[https://2008.igem.org/Team:Heidelberg/Notebook/material back]]
+===Plasmidvectors===
+{| class="wikitable"
+|- bgcolor=grey
+! height=20px, width=200px | Name || width=250px | application || width=250px | reference
+|-style="height:20px"
+|  ||  ||
+|-align="center"
+| style="font-weight:bold;" |[http://partsregistry.org/wiki/index.php?title=Part:BBa_B0015 BBa_B0015] || terminator || http://partsregistry.org
+|-align="center"
+| style="font-weight:bold;" |[http://partsregistry.org/wiki/index.php?title=Part:BBa_F1610 BBa_F1610] || LuxI || http://partsregistry.org
+|-align="center"
+| style="font-weight:bold;" |[http://partsregistry.org/wiki/index.php?title=Part:BBa_I15030 BBa_I15030] || AI-1 amplifier || http://partsregistry.org
+|-align="center"
+| style="font-weight:bold;" |[http://partsregistry.org/wiki/index.php?title=Part:BBa_I20260 BBa_I20260] || GFP || http://partsregistry.org
+|-align="center"
+| style="font-weight:bold;" |[http://partsregistry.org/wiki/index.php?title=Part:BBa_J01003 BBa_J01003] || oriT || http://partsregistry.org
+|-align="center"
+| style="font-weight:bold;" |[http://partsregistry.org/wiki/index.php?title=Part:BBa_J16002 BBa_J16002] || cloning vector || http://partsregistry.org
+|-align="center"
+| style="font-weight:bold;" |[http://partsregistry.org/wiki/index.php?title=Part:BBa_J23107 BBa_J23107] || constitutive promotor || http://partsregistry.org
+|-align="center"
+| style="font-weight:bold;" |[http://partsregistry.org/wiki/index.php?title=Part:BBa_T9002 BBa_T9002] || AI-1 GFP receiver || http://partsregistry.org
+|-align="center"
+| style="font-weight:bold;" |CheY-mCherry || cloning vector || V. Sourjik, ZMBH
+|-align="center"
+| style="font-weight:bold;" |ColE1 || colicin E1 || DSMZ
+|-align="center"
+| style="font-weight:bold;" |ColE9-J || colicin E9 || C. Kleanthous, University of York
+|-align="center"
+| style="font-weight:bold;" |pBad18 || cloning vector || V. Sourjik, ZMBH
+|-align="center"
+| style="font-weight:bold;" |pBad33 || cloning vector || V. Sourjik, ZMBH
+|-align="center"
+| style="font-weight:bold;" |pBluescript II SK (+) || cloning vector || V. Sourjik, ZMBH
+|-align="center"
+| style="font-weight:bold;" |pDest15  || cloning vector || DKFZ Library
+|-align="center"
+| style="font-weight:bold;" |pDK4 || visualization || V. Sourjik, ZMBH
+|-align="center"
+| style="font-weight:bold;" |pDK48 || cloning vector || V. Sourjik, ZMBH
+|-align="center"
+| style="font-weight:bold;" |pDK58 || cloning vector || V. Sourjik, ZMBH
+|-align="center"
+| style="font-weight:bold;" |pDK6 || visualization || V. Sourjik, ZMBH
+|-align="center"
+| style="font-weight:bold;" |pED374 || oriT || K. Derbyshire, Wadsworth
+|-align="center"
+| style="font-weight:bold;" |pES16 || cloning vector || V. Sourjik, ZMBH
+|-align="center"
+| style="font-weight:bold;" |pMMB863 || oriT || M. Bagdasarian, MSU
+|-align="center"
+| style="font-weight:bold;" |pQE-30 || cloning vector || Invitrogen
+|-align="center"
+| style="font-weight:bold;" |pSB1A2 || cloning vector || http://partsregistry.org
+|-align="center"
+| style="font-weight:bold;" |pSB2K3 || cloning vector || http://partsregistry.org
+|-align="center"
+| style="font-weight:bold;" |pTrc99a || cloning vector || V. Sourjik, ZMBH
+|-align="center"
+| style="font-weight:bold;" |pUB307 || helper plasmid || E. Lanka, BfR
+|-align="center"
+| style="font-weight:bold;" |RP4 || helper plasmid || M. Bagdasarian, MSU
+|-
+|}
+[[https://2008.igem.org/Team:Heidelberg/Notebook/material back]]
+===Synthetic oligonucleotides===
+All oligonucleotides were purchased from Invitrogen (Karlsruhe) and adjusted to a standard concentration of 100 <sup>pmol</sup>/<sub>µl</sub>.
+{| class="wikitable"
+|- bgcolor=grey
+! height=20px, width=250px | Name || width=450px | SEQUENCE (5´->3´)
+|-style="height:20px"
+|  ||
+|-align="center"
+| style="font-weight:bold;" |Bam_fw || GACAAGTGTTGGCCATGGAACAGG
+|-align="center"
+| style="font-weight:bold;" |Bam_rv || GCCGTCTGTGATGGCTTCCATG
+|-align="center"
+| style="font-weight:bold;" |cI_mut_fw || GCGTCTGGGTGGTGATGAGTTCACCTTCAAAAAACTG
+|-align="center"
+| style="font-weight:bold;" |cI_mut_rv || CAGTTTTTTGAAGGTGAACTCATCACCACCCAGACGC
+|-align="center"
+| style="font-weight:bold;" |CmR_EcoRI_mut_fw || GAATGCTCATCCGGAGTTCCGTATGGCAATG
+|-align="center"
+| style="font-weight:bold;" |CmR_EcoRI_mut_rev || CATTGCCATACGGAACTCCGGATGAGCATTC
+|-align="center"
+| style="font-weight:bold;" |CmR_fw || GCTAAAATGGAGAAAAAAATCACTGG
+|-align="center"
+| style="font-weight:bold;" |CmR_new_fw || TACGAGGTACCTTTACAGCTAGCTCAGTCCTAGGTATTATGC
+|-align="center"
+| style="font-weight:bold;" |CmR_new_rev || TATATAAGCTTTTACGCCCCGCCCTGCCACTCATCGCAGTACTGTTG
+|-align="center"
+| style="font-weight:bold;" |CmR_Prefix_fw || GAATTCGCGGCCGCTTCTAGAGTTTACAGCTAGCTCAGTCCTAGG
+|-align="center"
+| style="font-weight:bold;" |CmR_rv || AGGTTCTCCTTTATTAGCCGGATCCTCTAGATTACGCC
+|-align="center"
+| style="font-weight:bold;" |CmR_Suffix_rv || CTGCAGCGGCCGCTACTAGTATATAAACGCAGAAAGGCCCACCC
+|-align="center"
+| style="font-weight:bold;" |colE1_kil_prot_rv_A_SpeI || TATATACTAGTACTACTGAACCGCGATCCCCG
+|-align="center"
+| style="font-weight:bold;" |colE1_mut_EcoRI_fw || GGTATTGCTATTGTTACAGGTATTCTATGCTCCTATATTGATAAG
+|-align="center"
+| style="font-weight:bold;" |colE1_mut_EcoRI_rv || CTTATCAATATAGGAGCATAGAATACCTGTAACAATAGCAATACC
+|-align="center"
+| style="font-weight:bold;" |colE1_mut_PstI_1_fw || GCAGTAAAAGTGAAAGTTCAGCAGCTATTCATGCAACTGC
+|-align="center"
+| style="font-weight:bold;" |colE1_mut_PstI_1_rv || GCAGTTGCATGAATAGCTGCTGAACTTTCACTTTTACTGC
+|-align="center"
+| style="font-weight:bold;" |colE1_mut_PstI_2_fw || GCTGCCCGGGCAAAAGCAGCAGCGGAAGCACAGG
+|-align="center"
+| style="font-weight:bold;" |colE1_mut_PstI_2_rv || CCTGTGCTTCCGCTGCTGCTTTTGCCCGGGCAGC
+|-align="center"
+| style="font-weight:bold;" |colE1_mut_PstI_3_fw || CATTAGAGAAGAAAGCAGCAGATGCAGGGGTGAG
+|-align="center"
+| style="font-weight:bold;" |colE1_mut_PstI_3_rv || CTCACCCCTGCATCTGCTGCTTTCTTCTCTAATG
+|-align="center"
+| style="font-weight:bold;" |colE1_prot_fw_BamH1 || TACGAGGATCCATGGAAACCGCGGTAGCG
+|-align="center"
+| style="font-weight:bold;" |colE1_prot_rv_HindIII || TATATAAGCTTTTAAATCCCTAACACCTC
+|-align="center"
+| style="font-weight:bold;" |colE9_lysProt_rv_A_SpeI || TATATACTAGTACTAGGTTTTCGGCTTAGAACCCC
+|-align="center"
+| style="font-weight:bold;" |colE9_mut_EcoRI_fw || GTTGGGTGGACGATTCGAGAGTTCAATGGGGAAATAAAAATG
+|-align="center"
+| style="font-weight:bold;" |colE9_mut_EcoRI_rv || CATTTTTATTTCCCCATTGAACTCTCGAATCGTCCACCCAAC
+|-align="center"
+| style="font-weight:bold;" |colE9_plasmid_rv_A_SpeI || TATATACTAGTACACATGGAACTTTTGTGAC
+|-align="center"
+| style="font-weight:bold;" |colE9_prot_fw_BamH1 || TACGAGGATCCATCGATTTGCCCATGACCC
+|-align="center"
+| style="font-weight:bold;" |colE9_prot_rv_XmaI || TATATCCCGGGTTACTTACCTCGGTGAATATCG
+|-align="center"
+| style="font-weight:bold;" |DK13 || TCACCCGCACGCGC
+|-align="center"
+| style="font-weight:bold;" |DK167 || AGGATACTAGTAGGCCATTACTTT
+|-align="center"
+| style="font-weight:bold;" |DK9b || CGATGCGGCCGCTCAAAATGTTTCCCAGTTTGG
+|-align="center"
+| style="font-weight:bold;" |F1610_fw_XbaI || TACGATCTAGAAAAGAGGAGAAATACTAG
+|-align="center"
+| style="font-weight:bold;" |F1610_rv_HindIII || TATATAAGCTTTATAAACGCAGAAAGGCCC
+|-align="center"
+| style="font-weight:bold;" |GAM_fw || AGTGCTTTAGCGTTAACTTCCG
+|-align="center"
+| style="font-weight:bold;" |GAM_rv || GGTTTTACCGCATACCAATAACG
+|-align="center"
+| style="font-weight:bold;" |GFP_CmR_fw || CTCGTTGGTACCTCTAGATTTACAGCTAGCTCAGTCCTAGG
+|-align="center"
+| style="font-weight:bold;" |GFP_CmR_rv || TATTCGACCGGTACTAGTTATAAACGCAGAAAGGCCCACC
+|-align="center"
+| style="font-weight:bold;" |GFP_new_fw || TACGAGAGCTCTTTACAGCTAGCTCAGTCCTAGG
+|-align="center"
+| style="font-weight:bold;" |GFP_new_rv || TATATACCGGTACTAGTTATAAACGCAGAAAGGCCCACC
+|-align="center"
+| style="font-weight:bold;" |Lambda_insert_fw || TTGTAAAAACAGCCCTCCTC
+|-align="center"
+| style="font-weight:bold;" |Lambda_insert_rv || GATATGACTATCAAGGCCGC
+|-align="center"
+| style="font-weight:bold;" |LuxP_mut_F || CGTGAATTAGCAACAGAGTTCGGAAAGTTCTTCCC
+|-align="center"
+| style="font-weight:bold;" |LuxP_mut_R || GGGAAGAACTTTCCGAACTCTGTTGCTAATTCACG
+|-align="center"
+| style="font-weight:bold;" |LuxP_prefix_F || GAGGGAGAATTCGCGGCCGCTTCTAGATGAAGAAAGCGTTACTATTTTC
+|-align="center"
+| style="font-weight:bold;" |LuxP_sufffix_R || GGAGAGCTGCAGCGGCCGCTACTAGTAATTATCTGAATATCTAAATGCG
+|-align="center"
+| style="font-weight:bold;" |LuxPc || ATTACGCGGCCGCAGGAAACAGACCATGAAGAAAGCGTTACTATTTTCCC
+|-align="center"
+| style="font-weight:bold;" |LuxPd || GTAATGTCGACTCAATTATCTGAATATC
+|-align="center"
+| style="font-weight:bold;" |LuxP-seq-FW || CCCGTCCTGCCAGTGAGC
+|-align="center"
+| style="font-weight:bold;" |LuxQa || ATCGACCATGGGCAATAAATTTCGCTTAGC
+|-align="center"
+| style="font-weight:bold;" |LuxQc || GTAATGGATCCTTAGTGGAGGCTTGAGCC
+|-align="center"
+| style="font-weight:bold;" |LuxQTar_1a || CACAAATCATTGCCAATGAACGTATGTTGCTTACTCCGCTGG
+|-align="center"
+| style="font-weight:bold;" |LuxQTar_1b || CCAGCGGAGTAAGCAACATACGTTCATTGGCAATGATTTGTG
+|-align="center"
+| style="font-weight:bold;" |LuxQTar_2a || CTTAGCGACCATGAGCCATGAGTTTGCCCAGTGGCAACTGGC
+|-align="center"
+| style="font-weight:bold;" |LuxQTar_2b || GCCAGTTGCCACTGGGCAAACTCATGGCTCATGGTCGCTAAG
+|-align="center"
+| style="font-weight:bold;" |LuxS mutBbaIR || CCACACCCACTTCTAGGATGTTCTTCGCGATTTGC
+|-align="center"
+| style="font-weight:bold;" |LuxS mutXbaIF || GCAAATCGCGAAGAACATCCTAGAAGTGGGTGTGG
+|-align="center"
+| style="font-weight:bold;" |LuxS_mut_fw || CATCCTTTCTGAGAAAGGCATTCATACATTAGAGC
+|-align="center"
+| style="font-weight:bold;" |LuxS_mut_rev || GCTCTAATGTATGAATGCCTTTCTCAGAAAGGATG
+|-align="center"
+| style="font-weight:bold;" |LuxS_prefix_F || GGAGAGGAATTCGCGGCCGCTTCTAGATGGGCAATGCACCAGCGGTTCG
+|-align="center"
+| style="font-weight:bold;" |luxS_suffix_R || GAGGGACTGCAGCGGCCGCTACTAGTAGTCGATGCGTAGCTCTCTCAGC
+|-align="center"
+| style="font-weight:bold;" |LuxSa || ATCAGTCCATGGGCAATGCACCAGCGGTTCG
+|-align="center"
+| style="font-weight:bold;" |LuxSb || GTAATGGATCCTTAGTCGATGCGTAGC
+|-align="center"
+| style="font-weight:bold;" |mut_insert_fw || CTGAGGGGACGGTACCTCTACATTTACAGCTAGCTCAG
+|-align="center"
+| style="font-weight:bold;" |mut_insert_rv || CTGAGCTAGCTGTAAATGTAGAGGTACCGTCCCCTCAG
+|-align="center"
+| style="font-weight:bold;" |mut_insert2_fw || GGCAGGCGGGGCGTAATCTATAGGATCCGGCTAATAAAGG
+|-align="center"
+| style="font-weight:bold;" |mut_insert2_rv || CCTTTATTAGCCGGATCCTATAGATTACGCCCCGCCTGCC
+|-align="center"
+| style="font-weight:bold;" |mut_kpn1_pBlue || CGAGGGGGGGCCCGGTTCCCAATTCGCCCTATAG
+|-align="center"
+| style="font-weight:bold;" |mut_kpn1_pBlue || CTATAGGGCGAATTGGGAACCGGGCCCCCCCTCG
+|-align="center"
+| style="font-weight:bold;" |oriT_pre || GAATTCGCGGCCGCTTCTAGAGGACAGGCTCATGCCGGCCGC
+|-align="center"
+| style="font-weight:bold;" |oriT_RP4_fw || CTCGTTTCTAGAACTAGTGACAGGCTCATGCCGGCCGC
+|-align="center"
+| style="font-weight:bold;" |oriT_RP4_rv || TATTCGGGTACCGTCCCCTCAGTTCAGTAATTTCCTGC
+|-align="center"
+| style="font-weight:bold;" |oriT_suf || CTGCAGCGGCCGCTACTAGTAGTCCCCTCAGTTCAGTAATTTCCTGC
+|-align="center"
+| style="font-weight:bold;" |T9002_Lux_pR_rv_SpeI_BamHI_RBS || TATATACTAGTGGATCCGGTTCTGTTTCCTCTCTAGTATTTATTCGAC
+|-align="center"
+| style="font-weight:bold;" |T9002_LuxpR_Not_Eco_Xba_G_fw || TACGAGAATTCGCGGCCGCTTCTAGAGTCCCTATCAGTGATAGAGATTG
+|-align="center"
+| style="font-weight:bold;" |Term_new_fw || TACGAAAGCTTCCAGGCATCAAATAAAACGAAAGG
+|-align="center"
+| style="font-weight:bold;" |Term_new_rv || TATATGAGCTCTATAAACGCAGAAAGGCCCACCC
+|-align="center"
+| style="font-weight:bold;" |VF2 || TGCCACCTGACGTCTAAGAA
+|-align="center"
+| style="font-weight:bold;" |VIC121 || TTTATCGCAACTCTCTACTG
+|-align="center"
+| style="font-weight:bold;" |VIC122 || CTGATTTAATCTGTATCAGG
+|-align="center"
+| style="font-weight:bold;" |VIC131 || ATGTGTGGAATTGTGAGCGG
+|-align="center"
+| style="font-weight:bold;" |VIC132 || CTGATTTAATCTGTATCAGG
+|-align="center"
+| style="font-weight:bold;" |VR || ATTACCGCCTTTGAGTGAGC
+|-
+|}
+[[https://2008.igem.org/Team:Heidelberg/Notebook/material back]]
+===Phages===
+{| class="wikitable"
+|- bgcolor=grey
+! height=20px, width=200px | Name || width=250px | application || width=250px | reference
+|-style="height:20px"
+|  ||  ||
+|-align="center"
+| style="font-weight:bold;" |Lambda cI mut || cI deleted || W. Reiser, ZMBH
+|-align="center"
+| style="font-weight:bold;" |Lambda cI857 || heat inducible || MBI Fermentas
+|-
+|}
+[[https://2008.igem.org/Team:Heidelberg/Notebook/material back]]
+===Bacteria===
+{| class="wikitable"
+|- bgcolor=grey
+! height=20px, width=200px | E.coli strain || width=250px | usage || width=250px | reference
+|-style="height:20px"
+|  ||  ||
+|-align="center"
+| style="font-weight:bold;" |DH5a || amplification of plasmids || Invitrogen
+|-align="center"
+| style="font-weight:bold;" |HCB33 || swarm assays || V. Sourjik, ZMBH
+|-align="center"
+| style="font-weight:bold;" |MG1655 || swarm assays || V. Sourjik, ZMBH
+|-align="center"
+| style="font-weight:bold;" |TOP10 || amplification of plasmids || Invitrogen
+|-align="center"
+| style="font-weight:bold;" |UU1250 || chemotaxis receptor knock out || V. Sourjik, ZMBH
+|-
+|}
+[[https://2008.igem.org/Team:Heidelberg/Notebook/material back]]
+===Bacteria Growth Media===
+{| class="wikitable"
+|- bgcolor=grey
+|-align="center"
+| style="font-weight:bold;" |Luria Broth (LB) || 10 <sup>g</sup>/<sub>l</sub> || tryptone
+|-align="center"
+| style="font-weight:bold;" | || 5 <sup>g</sup>/<sub>l</sub> || yeast extract
+|-align="center"
+| style="font-weight:bold;" | || 10 <sup>g</sup>/<sub>l</sub> || NaCl
+|-align="center"
+| style="font-weight:bold;" | || ||
+|-align="center"
+| style="font-weight:bold;" |Luria Broth (LB) plus || 10 <sup>g</sup>/<sub>l</sub> || tryptone
+|-align="center"
+| style="font-weight:bold;" | || 5 <sup>g</sup>/<sub>l</sub> || yeast extract
+|-align="center"
+| style="font-weight:bold;" | || 20 <sup>g</sup>/<sub>l</sub> || NaCl
+|-align="center"
+| style="font-weight:bold;" | || ||
+|-align="center"
+| style="font-weight:bold;" |TB || 10 <sup>g</sup>/<sub>l</sub> || bacto tryptone
+|-align="center"
+| style="font-weight:bold;" | || 5 <sup>g</sup>/<sub>l</sub> || NaCl
+|-align="center"
+| style="font-weight:bold;" | || ||
+|-align="center"
+| style="font-weight:bold;" |Standard I || 15.6 <sup>g</sup>/<sub>l</sub> || peptone P
+|-align="center"
+| style="font-weight:bold;" | || 2.8 <sup>g</sup>/<sub>l</sub> || yeast extract
+|-align="center"
+| style="font-weight:bold;" | || 5.6 <sup>g</sup>/<sub>l</sub> || NaCl
+|-align="center"
+| style="font-weight:bold;" | || 1 <sup>g</sup>/<sub>l</sub> || D-glucose
+|-align="center"
+| style="font-weight:bold;" | || ||
+|-align="center"
+| style="font-weight:bold;" |Minimal medium || 9.8 % || M63 salts
+|-align="center"
+| style="font-weight:bold;" | || 0.2 %|| glycerol
+|-align="center"
+| style="font-weight:bold;" | || 0.1 <sup>g</sup>/<sub>l</sub> || Thiamine
+|-align="center"
+| style="font-weight:bold;" | || 1 mM || MgSO<sub>4</sub>
+|-align="center"
+| style="font-weight:bold;" | || 0.8 % || amino acid mix
+|-align="center"
+| style="font-weight:bold;" | || ||
+|-align="center"
+| style="font-weight:bold;" |M9 || 20 % || M9 salts
+|-align="center"
+| style="font-weight:bold;" | || 2 mM || MgSO<sub>4</sub>
+|-align="center"
+| style="font-weight:bold;" | || 0.4 %|| glucose
+|-align="center"
+| style="font-weight:bold;" | || 0.1 mM || CaCl<sub>2</sub>
+|-align="center"
+|-
+|}
+For cultivation of phages the media were supplied with 0.2 % maltose and 10 mM MgSO4. For preparation of agar plates 15 g/l agar, for preparation of top agar 7 g/l agar was added prior the autoclaving. For selection of resistant bacteria following antibiotics were added during cooling of at agar at about 50 °C:
+[[https://2008.igem.org/Team:Heidelberg/Notebook/material back]]
+{| class="wikitable"
+|- bgcolor=grey
+! height=20px, width=200px | Antibiotic || width=250px | concentration
+|-style="height:20px"
+|  ||
+|-align="center"
+| style="font-weight:bold;" |Ampicillin || 100 µg/ml
+|-align="center"
+| style="font-weight:bold;" |Chloramphenicol || 35 µg/ml
+|-align="center"
+| style="font-weight:bold;" |Kanamycin || 50 µg/ml
+|-
+|}
+The same concentrations of antibiotics were used for selection of resistant in media.
+[[https://2008.igem.org/Team:Heidelberg/Notebook/material back]]
+==Methods==
+===Preparing chemically competent cells===
+First, a 20 ml over night culture was inoculated in antibiotic free LB medium from a fresh single colony and transferred into 400 ml antibiotic free LB medium the next day. This culture was incubated at 37 °C while shacking until an OD600 of 0.5 – 0.6 was achieved. The culture was than cooled down on ice, centrifuged (8 min, 4 °C, 3500 rpm), the supernatant discarded and the pellet resuspended in 10 ml 100 mM CaCl<sub>2</sub>. After addition of further 190 ml 100 mM CaCl<sub>2</sub> the suspension was incubated on ice for 30 min. The suspension was than again centrifuged (8 min, 4 °C, 3500 rpm), the supernatant discarded, the pellet resuspended in 20 ml 82.5 mM CaCl2 with 17.5 % glycerol and aliquoted. The aliquots were flash frozen in liquid nitrogen and than stored at -80 °C until usage.
+[[https://2008.igem.org/Team:Heidelberg/Notebook/material back]]
+===Transformation of bacteria===
+For enrichment of vectors, ''E .coli'' TOP10 and DH5α were used. For the transformation 100 µl of the competent cells were thawed on ice and 50 – 200 ng DNA solution added (depending on the concentration of the DNA solution). After a 20 minute incubation on ice, cells were made permeable for the DNA by heat shocking for 45 seconds at 42 °C and a further 2 minute incubation on ice. The samples were than rescued by adding 250µl preheated antibiotic free LB-medium and incubated for one hour at 37 °C while shacking for induction of the antibiotic resistance. The selection for plasmid containing and therefore antibiotic resistant bacteria was conducted by plating them on antibiotic containing LB-agar plates.
+[[https://2008.igem.org/Team:Heidelberg/Notebook/material back]]
+===Isolation of plasmid DNA by alkaline lysis (mini and maxiprep)===
+For analysis of ligations and transformations QIAprep Spin Kits (Qiagen, Hilden) were used following the manufacturer instructions.
+For miniprep a single colony was picked from a LB-agar plate or glycerol stock was used to inoculate 5 ml LB-medium with appropriate antibiotic for selection (100 µg/µl ampicillin, 50 µg/µl kanamycin, 35 µg/µl chloramphenicol). Bacteria were grown over night at 37 °C while shaking (200 rpm). By using 4 ml over night culture with this kit the yield was around 6-10 µg.
+For maxipreps the Qiagen CompactPrep Plasmid Maxi Kit was used following the instructions given by the instruction manual. In this case 250 ml LB-medium were inoculated and used for preparation of plasmid DNA. The routinely yield was 300-400 µg plasmid DNA. Purity and amount of DNA was analysed using a NanoDrop.
+[[https://2008.igem.org/Team:Heidelberg/Notebook/material back]]
+===DNA amplification using polymerase chain reaction (PCR)===
+By using PCR smallest amount of DNA can be detected and amplified. The principle of PCR is the selective amplification of any region of the DNA. Nevertheless, the sequence at both ends must be known for the binding of two complementary primers. The DNA region of interest is than amplified exponentially while the reproduction of the DNA takes place in three temperature stpes: denaturation of parental DNA at high temperature (95-98 °C), hybridisation of primers (58-65 °C) and DNA elongation (72 °C).
+For the amplification a heat-stable DNA polymerase I with a 3’-5’ exonuclease activity (proof reading) is used such as Pfu (from Pyrococcus furiosus) and Phusion (a Pyrocuccus-like enzyme) or the non proof reading enzyme Taq (from Thermus aquaticus).
+Phsuion polymerase was used in a 2x master mix adding only primers (20pmol each) and DNA template (~10ng) or alternatively colonies from agar plates (1-5 colonies) in a final volume of 50 µl. For taq and Pfu polymerase following reaction batch was commonly used:
+{| class="wikitable"
+|- bgcolor=grey
+! height=20px, width=900px |
+|-align="center"
+| style="font-weight:bold;" | 1 µl   template (10 ng) / colonies
+|-align="center"
+| style="font-weight:bold;" | 2 µl   primer 1 (10 pmol/µl)
+|-align="center"
+| style="font-weight:bold;" | 2 µl   primer 2 (10 pmol/µl)
+|-align="center"
+| style="font-weight:bold;" | 1 µl   polymerase (2,5 Units)
+|-align="center"
+| style="font-weight:bold;" | 1 µl   dNTP mix (10 mM)
+|-align="center"
+| style="font-weight:bold;" | 5 µl   10x buffer
+|-align="center"
+| style="font-weight:bold;" |38 µl   dH2O
+|-
+|}
+The PCR procedure was as follows:
+{| class="wikitable"
+|- bgcolor=grey
+! height=20px, width=300px | || width=300px| || width=300px|
+|-align="center"
+| style="font-weight:bold;" |Initiale denaturation || 95- 98 °C, 3-5 min || 1 cycle
+|-style="height:20px"
+|
+|-align="center"
+| style="font-weight:bold;" |denaturation || 95-98 °C, 15 sec - 1 min
+|-align="center"
+| style="font-weight:bold;" |annealing || 58-65 °C, 15 sec - 1 min || 25-28 cycles
+|-align="center"
+| style="font-weight:bold;" |elongation || 72 °C, 15 sec - 3 min
+|-style="height:20px"
+|
+|-align="center"
+| style="font-weight:bold;" |termination || 72 °C, 5 – 10 min || 1 cycle
+|-align="center"
+| style="font-weight:bold;" | || 4 °C || forever
+|-style="height:20px"
+|
+|-
+|}
+For site directed mutagenesis PCR Pfu turbo polymerase (Stratagene) was used in the same reaction batch described above. The temperature program was as follows:
+{| class="wikitable"
+|- bgcolor=grey
+! height=20px, width=300px | || width=300px| || width=300px|
+|-align="center"
+| style="font-weight:bold;" |initiale denaturation || 95 °C, 30 sec || 1 cycle
+|-style="height:20px"
+|
+|-align="center"
+| style="font-weight:bold;" |denaturation || 95 °C, 30 sec
+|-align="center"
+| style="font-weight:bold;" |annealing || 55 °C, 1 min || 16 cycles
+|-align="center"
+| style="font-weight:bold;" |elongation || 68 °C, 2 min per kb of plasmid
+|-style="height:20px"
+|
+|-align="center"
+| style="font-weight:bold;" |termination || 68 °C, 10 min || 1 cycle
+|-align="center"
+| style="font-weight:bold;" | || 4 °C || forever
+|-style="height:20px"
+|
+|-
+|}
+For point mutagenesis primers with around 33 bases were used having the base to be altered in the middle. The melting temperature of these primers was always over 78 °C. If this melting temperature could not be achieved using 15 unchanged nucleotides at both sites the flanking arms were enlarged properly. After the PCR reaction 10 units of DpnI was added directly to the PCR tube, mixed and incubated for 1-5 h at 37 °C. DpnI digests the parental methylated plasmid DNA leaving only the mutated one. After purification using QIAquick PCR Purification Kit the plasmid was transformed as described above.
+[[https://2008.igem.org/Team:Heidelberg/Notebook/material back]]
+===Purification of DNA from PCR reactions===
+PCR products were purified by the QIAquick PCR Purification Kit from Qiagen following the instructions of the Qiagen Handbook. To check the purity and amount of extracted DNA an aliquot was analysed using a NanoDrop.
+[[https://2008.igem.org/Team:Heidelberg/Notebook/material back]]
+===Enzymatic hydrolysis of DNA by restriction enzymes===
+The restriction digest of DNA was used for analysis of purified DNA form mini or maxiprep or for isolation of specific DNA fragments for further cloning. Analytical digestions were routinely conducted in 20 µl volume. In all digestions a minimum of 2 Units restriction enzyme(s) was used per microgram DNA. Optimised buffer conditions were secured by using NEB buffer system. The final reaction volume was achieved by adding H<sub>2</sub>O dest. and the sample was incubated at optimal temperature for the restriction enzyme(s) (normally 37 °C). Preparative digestions were conducted in a volume of 50 µl. For analysis and preparation DNA loading dye was added to the samples and they were loaded on a agarose gel or alternatively purified by QIAquick PCR Purification Kit from Qiagen.
+[[https://2008.igem.org/Team:Heidelberg/Notebook/material back]]
+===Agarose gel electrophoresis for separating DNA===
+In the agarose gel electrophoresis a mixture of DNA fragments with different sizes are separated in an electrical field by their size. This is achieved by moving the negatively charged DNA through an agarose matrix while shorter fragments will run faster. The size of the pores can be controlled by agarose concentration. The higher the agarose concentration the smaller the pores are and the smaller fragments can be separated. Agarose concentrations between 0.7 and 1.5 % agarose in 0.5x TE buffer were used.
+The agarose was dissolved completely by heating up and 0.1 µg/ml ethidium bromide was added.
+The DNA fragments were separated using a constant voltage between 80 and 130 V. Under UV light (λ = 254 nm) DNA is visible through the unspecific intercalated ethidium bromide and can be documented or cut out and extracted from the gel.
+[[https://2008.igem.org/Team:Heidelberg/Notebook/material back]]
+===Isolation of DNA fragments from an agarose gel===
+Plasmid DNA and DNA fragments were extracted using the Gel Extraction Kit from Qiagen following the manufacture instructions. To check the purity and amount of extracted DNA an aliquot was analysed using a NanoDrop.
+[[https://2008.igem.org/Team:Heidelberg/Notebook/material back]]
+===Ligation of dsDNA fragments===
+T4 DNA ligase was used to form covalent phosphodiesterbonds between doublestranded DNA fragments having blunt or compatible sticky ends. For the ligation the restricted vector -DNA and the insert were mixed 1:3 or 1:5 in a total ligationvolume of 20 µl. This mixture was incubated in ligation buffer using 5 Weiss units T4 DNA ligase over night at 16 °C or at room temperature for 20 minutes and afterwards used to transform chemical competent cells.
+[[https://2008.igem.org/Team:Heidelberg/Notebook/material back]]
+===Lysing of bacteria by ultrasonication===
+For the lysis of bacteria an ultrasonic tip was used. The 10-15 ml of bacteria over night cultures were sonicated with an ultrasonic tip three time for 15 seconds. After sonication cell extract was observed under microscope.
+[[https://2008.igem.org/Team:Heidelberg/Notebook/material back]]
+===Glycerol stock===
+To store bacteria for long term glycerol stocks were used. Therefore 1 ml of an over night culture were added to 150 µl of 80 % Glycerol into a cryo tube, vortexed and incubated at room temperature for 30 min. Afterwards the glycerole stock was stored at -80 °C.
+[[https://2008.igem.org/Team:Heidelberg/Notebook/material back]]
+===Preparation of plating bacteria for bacteriophage experiments===
+A overnight culture of the appropriate ''E. coli'' strain was grown in LB medium containing 10 mM MgSO<sub>4</sub> and 0.2 % maltose at 30 °C to reduce the amount of cell debris in the medium. The added maltose leads to a substantial induction of the maltose operon including the lamb gene, which encodes the cell surface receptor to which bacteriophage λ binds. After harvesting the cells they were resuspended in 10 mM MgSO<sub>4</sub> and diluted to a final concentration of 2.0 OD<sub>600</sub>. The suspension of plating bacteria was stored at 4 °C for up to 1 week.
+[[https://2008.igem.org/Team:Heidelberg/Notebook/material back]]
+===Bacteriophage λ plaque assay===
+Tenfold serial dilutions of the bacteriophage stocks were prepared, 100 µl of it mixed with the same amount of plating bacteria, incubated for 20 minutes at 37 °C to allow the bacteriophage particles to adsorb to the bacteria, this mixture added to 3 ml molten top agar which was kept liquid at 48 °C and the entire contend poured onto a agar plate. After harden of the top agar the inverted plates were incubated at 37 °C over night. On the next day plaques could be counted.
+[[https://2008.igem.org/Team:Heidelberg/Notebook/material back]]
+===Preparing Stocks of Bacteriophage λ===
+Completely lysed agarose plates were prepared by mixing 0.1 ml plating bacteria with ~10<sup>5</sup> pfu, incubation for 20 minutes at 37 °C and that bringing out the mixture in 3 ml molten top agarose. Agarose is preferred in this protocol in order to minimize contaminations with polyanionic inhibitors that can interfere with subsequent enzymatic analysis of the bacteriophage DNA. The plates were incubated 12-16 hours without inversion at 37 °C. Plates were not inverted during incubation to encourage sweating of fluid onto the surface of the dish, which allows the bacteriophage to spread more easily. The plates were than overlayed with 5 ml SM and incubated while shaking at 4 °C over night. The SM was that transferred to a tube and the plate washed again with 1 ml SM. 2 % v/v chloroform was added and bacterial debris removed by centrifugation at 10,000 g for 10 minutes at 4 °C. The concentration of infectious particles in the stock was measured by plaque assays as described above.
+[[https://2008.igem.org/Team:Heidelberg/Notebook/material back]]
+===Preparing of λ DNA===
+Lambda DNA was prepared using the QIAGEN® Lambda mini kit following the manufacture instructions.
+[[https://2008.igem.org/Team:Heidelberg/Notebook/material back]]
+===In vitro packaging of λ DNA===
+In vitro packaging was conducted using the MaxPlax lambda packaging extracts from Epicentre Biotechnologies following the manufacture instructions.
+[[https://2008.igem.org/Team:Heidelberg/Notebook/material back]]
+===Colicin Activity Test===
+To measure the killing efficiency and which amount of cells or colicins are needed to reach any killing activity a colicin activity test was carried out. Therefore bacteria containing the colicin plasmid (''E. coli'' TOP10 or ''E. coli'' MG1655) and GFP producing cells (reference promoter [http://partsregistry.org/wiki/index.php?title=Part:BBa_I20260 BBa_I20260], ''E. coli'' TOP10) were inoculated in TB-media with appropriated antibiotics at 37 °C for 4 to 6 hours and the optical densities of the two strains were adapted. The colicin cells themselves, their produced supernatant or the supernatant of the lysed colicin cells were added in different ratios to a constant amount of GFP producing cells. The total volume was kept constant and the missing amount added with TB-media without antibiotics. The colicin production was induced by several concentrations (0 M-100 nM) of N-Acyl-Homoserin-Lactone (AHL). The OD and GFP intensities were measured at 37 °C in the Tecan Microplate Reader every half an hour for about 12 hours.
+As a negative control the similar test was carried out with cells, containing the same plasmid without the colicin operon on it.
+[[https://2008.igem.org/Team:Heidelberg/Notebook/material back]]
+===Lysis test===
+Lysis of killercells dependent on the AHL concentration was measured due to a lysis test. Therefore ''E. coli'' TOP 10 containing the luxR-colicinE1 part ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K150009 BBa_K150009]) and a GFP producing part ([http://partsregistry.org/wiki/index.php?title=Part:BBa_I20260 BBa_I20260]) were inoculated in TB-media with appropriate antibiotics at 37 °C for 4 to 6 hours. As reference ''E. coli'' TOP 10 cells containing a luxR part (comparable to [http://partsregistry.org/wiki/index.php?title=Part:BBa_F2620 BBa_F2620]) instead of the luxR-colicinE1 part ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K150009 BBa_K150009]). For the lysis measurement 250 µl of the liquid cultures were diluted with 250 µl TB-media. The Lux pR promoter was activated with AHL concentrations from 0 M up to 50 nM. The optical densities and GFP intensities were measured in Tecan Microplate Reader every half-hourly for about 12 hours.
+[[https://2008.igem.org/Team:Heidelberg/Notebook/material back]]
+===Sender Activity Test===
+The sender activity test was performed to quantify the AHL production of the cells containing the sender part ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K150000 BBa_K150000]). Therefore TB-media with the appropriated antibiotic was inoculated with 2 µl/ml of an overnight culture. For 7 hours every hour the OD of the medium was measured and 5 ml of the culture were spin down to produce supernatant. The sterile filtered supernatant, still containing the AHL, was stored at 4 °C. The supernatant of every hour was then added in different ratios to a constant amount of AHL inducible cells which produce GFP after induction. The OD and GFP intensities were measured at 37 °C in the Tecan Microplate Reader every half an hour for about 12 - 24 hours. The same test was carried out with amplifier cells ([http://partsregistry.org/wiki/index.php?title=Part:BBa_I15030 BBa_I15030]). To calculate the produced amount of AHL by sender or amplifier cells as reference the test were performed also with different AHL concentration (0 M-100 nM) instead of AHL producing cells.
+[[https://2008.igem.org/Team:Heidelberg/Notebook/material back]]
+===LuxS Activity Test===
+This protocol for the functional check AI-2 production is mainly adapted from a paper of Vanderleyden et al. [1] , for further questions please refer to it. 10ml ONC DH5α strain with LuxS (S) and 10ml DH5α strain with ptrc99α as blank (B) were incubated in LB to a volume of 50mL each at 37°C up to an OD of 0.35 in 100ml erlenmeyer flasks. Then S and B each were induced with 500 µM IPTG for 3h, following centrifugation at maximum speed for 20min. The supernatants were then transferred in two new falcons by pipetting or decanting. Optional the supernatant was sterile filtrated and/or 10 g/L NaCl was added to a final concentration of ~20g/L. 10 mL ONC of ''V. harveyi'' reporter (BB120 in LB plus at 30 °C) was incubated in LB plus to a final volume of 50 mL at 37°C up to an OD of 0.35 in a 100 mL erlenmeyer flask. Supernatant S and B were both mixed with ''Vibrio'' cells at ratios 1:3, 1:5 and 1:10 each to a final volume of 5 mL in 50 mL falcon tubes and incubated for 30min at 30°C. Subsequently the cells were incubated again for 3h, OD and Luminescence were measured with a microplate reader. [[https://2008.igem.org/Team:Heidelberg/Notebook/material back]]
+[1] Sigrid C. J. DeKeersmaecker and Jos Vanderleyden ‘Constraints on detection of autoinducer-2 (AI-2) signalling molecules using Vibrio harveyi as a reporter’, Microbiology 2003 149: 1953-1956.
+===Conjugation test===
+Equal amounts of donor (cells harbouring the oriT containing plasmid and pUB307) and acceptor cells from overnight cultures were centrifuged (2 min, 13000 rpm), the pellet washed twice with 1.5 ml LB medium without antibiotics, the pellets combined and this mixture resuspended in 100 µl LB medium without antibiotics. This cell mix was transferred to a filter membrane on a LB agar plate and incubated here for a time range from 0 to 42 minutes in 6 minutes steps at 37 °C. The membrane was then transferred in 1 ml LB medium, vortexed, the medium diluted and plated out on LB agar plates with proper antibiotics for plasmid harbouring the oriT and antibiotic resistance of acceptor bacteria. On the next day the number of transconjugants was counted and therewith the conjugation efficiency calculated as ratio of cell density of transconjugants to cell density of donor cells depending on the reaction time.
+[[https://2008.igem.org/Team:Heidelberg/Notebook/material back]]
+=== Receptor Polarization Test ===
+To check the functionality of the fusion receptor constructs each construct, on pDK48, was transformed into strain UU1250 together with pDK36 (YFP-CheA). The expression of the receptor was induced with 0.01% arabinose, of YFP-CheA with 30µM IPTG. Polarization was tested by putting 10 µL of the cells on a 0.5% agarose pad and making snapshots using a Zeiss Axio Imager.Z1 fluorescent microscope.
+===Swarm Assays===
+The sensitivity of the LuxP-tar fusion constructs to AI-2 was tested in swarm assays. For this two different strains were used: ''E. coli'' UU1250 and ''E. coli'' HCB33. The assays were performed in TB medium for UU1250 and TB or minimal medium for HCB33 (0.3% agar). As chemo attractant either 3 µl of an AI-2 producing ONC, induced with 100 nM IPTG, or 10 µl supernatant from those cells was spotted 13 times on the plate to create a center line. Gradient building was allowed for 12-16 hours at 4 °C. Afterwards the fusion receptor expressing cells (0.01% arabinose induced overnight) as well as non-chemotactic reference strains were spotted at a distance of about 3-3.5 cm to the attractant line. Swarm plates were incubated at 30 °C and pictures taken after 24, 48 and 72 hours.
+[[https://2008.igem.org/Team:Heidelberg/Notebook/material back]]
+===Microscopy – Chemotaxis===
+An Applied Precision - DeltaVision wide field microscope was used to visualize chemotactic behaviour of the LuxP-tar fusion. ''E. coli'' UU1250 and ''E. coli'' HCB33 were used for these measurements. The monitoring was performed with Labtek<sup>TM</sup> chambered coverglass systems (Nunc) which were filled with 2 ml of TB media, Tethering Buffer and minimal media containing 0.2 % agar. As chemo attractant either 3 µl of an AI-2 producing ONC, induced with 100 nM IPTG, or 10 µl supernatant from those cells was spotted at the one side of the chamber. Gradient building was allowed for 12-16 hours at 4 °C.
+Afterwards either fusion receptor expressing cells (0.01% arabinose induced overnight) or non-chemotactic reference strains were spotted at the center of the chamber. The chamber was then incubated for several hours at 30 °C. After 3 hours frame pictures were taken, using confocal fluorescence imaging and 8x6 frames. (8 pictures in length, 6 in height)
+Images were processed and auto arranged with housemade ImageJ plugins. Matlab was used for the quantitative analysis.
+[[https://2008.igem.org/Team:Heidelberg/Notebook/material back]]
+===Microscopy – Colicin Toxicity===
+The killing efficiency of colicin producing cells was monitored using an Applied Precision - DeltaVision wide field microscope. Therefore a prey and a killer strain were designed in ''E. coli'' TOP10. The prey strain contained a GFP producing part ([http://partsregistry.org/wiki/index.php?title=Part:BBa_I20260 BBa_I20260]) as well as the luxI sender part ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K150000 BBa_K150000]) and the killer strain contained as marker a mCherry producing part as well as the LuxR-colicinE1-receiver ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K150009 BBa_K150009]).
+Measurements were prepared with 1 ml of a prey ONC and 1 ml of a killer ONC in 3 ml TB media containing 0,01% arabinose and 10 nM AHL for induction of colicin production. A second probe in which cells without colicin genes were used instead of the killer cells was prepared in parallel. Both samples were incubated for 6-7 hours at 37 °C.
+Monitoring was performed on the Deltavision microscope with 8-well Labtek<sup>TM</sup> chambered coverglass systems (Nunc). For that 6 µl of each sample were distributed equaly in 200 µl TB media. Frame pictures have been taken using 8x8 frames evenly distributed over the whole chamber.
+Images were processed and auto arranged with housemade ImageJ plugins.
+[[https://2008.igem.org/Team:Heidelberg/Notebook/material back]]
+===Microscopy – Cell lysis===
+To visualize the lysis of prey and killer cells cultures were observed on Zeiss Axio Imager.Z1 microscope. The killer strain (''E. coli'' TOP 10) contained the luxR-colicinE1 part ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K150009 BBa_K150009]) and a mCherry producing part and the prey strain contained the senderpart ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K150000 BBa_K150000]) and a GFP producing part ([http://partsregistry.org/wiki/index.php?title=Part:BBa_I20260 BBa_I20260]). Both strains were inoculated in TB-media with appropriate antibiotics at 37 °C for 4 to 6 hours. Thereafter OD was adjusted and the killer strain was induced by 25 nM AHL. After 2 hour 200 µl of the induced killer culture and 200 µl of the prey culture were combined and centrifuged down. For microscopy pelleted cells were diluted with 10 µl and plated on a 0.5% agarosepad. Fixed positions were observed half hourly in the mCherry and GFP channel with a 100x oil immersion objective.
+[[https://2008.igem.org/Team:Heidelberg/Notebook/material back]]
+===Cell Culture===
+MCF-7 cells (breast cancer cell line) wt or stably expressing cytoplasmic GFP, were plated in 24 well plates (150 thousand cells per well) with 10 % FCS (fetal calf serum) DMEM (Dulbecco's Modified Eagle Medium) and incubated overnight at 37 °C, 5 % CO2.
+E. coli TOP 10 strains containing LuxR-colicin E1-receiver (BBa_K150009) and strains containing LuxR-receiver (no colicin) were grown overnight in TB media with appropriate antibiotics at 37 °C to an OD of 1.0 and diluted in the morning to 0.1. Once bacterial cells reached an OD of 0.35 they were harvested and inoculated on the MCF-7 wt and on MCF-7-GFP cells.
+[[https://2008.igem.org/Team:Heidelberg/Notebook/material back]]
+===Eucaryotic Killing Assay with Colicins===
+Once MCF-7 cells were confluent (12h after passage), E. coli TOP 10 bacteria were spinned down (1 ml of OD = 0.35) and resuspended with DMEM medium from the original wells containing the MCF-7 wt/MCF-7-GFP cells. A 24-well plate was used containing MCF-7  cells stained with Propidium Iodide (PI) alone, with LuxR-receiver cells not containing colicin and LuxR-receiver-colicin E1 cells. The second 24-well plate with MCF-7GFP was processed as described for wt MCF-7, but without any PI staining. AHL was added to every well to a final concentration of 5 nM. Well content was collected at different time-points (0 – 6 h) and when necessary, PI was added to the suspension 15 minutes before Flow Cytometer analysis.
+[[https://2008.igem.org/Team:Heidelberg/Notebook/material back]]
+===Flow Cytometer Acquisition===
+At the referred timepoints, supernatants were collected and spinned down. Eukaryotic cells were detached by trypsinization (200 μl) for 10 minutes at 37 °C and then resuspended in 800 μl of KH + 1 % BSA (Krebs Henseleit medium with 1 % Bovine Serum Albumin). After resuspending, well content was added to the precipitated supernatant to account for floating dead cells. PI was added to each sample (1:1000) and incubated at RT for 15 minutes before acquisition. PI positive cells and GFP expressing cells (%) were measured for 3000 cells for each condition. Histograms and fluorescent measurements were obtained using a Cytomics FC 500 MPL from Beckman&Coulter.
+[[https://2008.igem.org/Team:Heidelberg/Notebook/material back]]

Team:Heidelberg/Notebook/material

From 2008.igem.org

Latest revision as of 09:15, 20 August 2009

Contents

Material

Buffers & Solution

Kits

Marker

Enzymes

Plasmidvectors

Synthetic oligonucleotides

Phages

Bacteria

Bacteria Growth Media

Methods

Preparing chemically competent cells

Transformation of bacteria

Isolation of plasmid DNA by alkaline lysis (mini and maxiprep)

DNA amplification using polymerase chain reaction (PCR)

Purification of DNA from PCR reactions

Enzymatic hydrolysis of DNA by restriction enzymes

Agarose gel electrophoresis for separating DNA

Isolation of DNA fragments from an agarose gel

Ligation of dsDNA fragments

Lysing of bacteria by ultrasonication

Glycerol stock

Preparation of plating bacteria for bacteriophage experiments

Bacteriophage λ plaque assay

Preparing Stocks of Bacteriophage λ

Preparing of λ DNA

In vitro packaging of λ DNA

Colicin Activity Test

Lysis test

Sender Activity Test

LuxS Activity Test

Conjugation test

Receptor Polarization Test

Swarm Assays

Microscopy – Chemotaxis

Microscopy – Colicin Toxicity

Microscopy – Cell lysis

Cell Culture

Eucaryotic Killing Assay with Colicins

Flow Cytometer Acquisition