Team:Goettingen/NoteBook w0

Selection of clones from transformation of pGP172 + cdaA (L. monocytogenes) and pGP172 + DAC domain (L. monocytogenes)

pGP172 + cdaA

1 colony on 100 μl plate 14 clones on concentrated plate

→ it may be presummed that the ful length protein is toxic!

→ 10 clones were selected named: #81 - #90

pGP172 + DAC domain

many clones on 100 μl plate many clones on concentrated plate

→ re-streak them on LB Amp100 plates and incubate for 5 h at 37°C; store them for later use at 4°C

→ inoculate 4 ml LB Amp100 liquid media over night at 37°C and spread them afterwards on LB Amp100 plates

→ 10 clones were selected and named: #101- #110

 

Cololny PCR of selected clones for pGP172 + cdaA (L. monocytogenes) and pGP172 + DAC domain (L. monocytogenes)

The selected clones were analyzed by colony PCR

Components	Volume(μl)
Forward primer (T7 promoter) [5μM]	1
Reverse primer (iGEM 9) [5μM]	2
dNTPs [12.5 μM]	1
10x Taq buffer	2.5
Taq polymerase	0.5
dd H2O	18
Total	25

 

Transformation of pGP172 + cdaA (L. monocytogenes) and pGP172 + DAC domain (L. monocytogenes)

Protocol:

- thaw 200 μl of competent E. coli cells on ice

- add ligation mixture to the cells and mix carefully

- incubate the transformation mixture for 30 min on ice

- transfer the samples for 90 sec to 42°C (heat shock)

- replace samples for 5 min to ice and ad 800 μl liquid LB medium incubate transformation mixture for 1 h at 37°C and agitation

-add 100 μl of the mixure onto LB Amp100 media plates

- centrifuge the samples for 1 min at 13,000 rpm, discard the upper supernatant ans resuspend cells in the remaining 100 μl

-plate concentrated cells as well on LB Amp100 medium plates

- incubate plates overnight at 37°C - store plates afterwards at 4°C

 

 

 

Creation of Agar plates

-          Inoculation of 2 x 250 ml CSE – Glc medium with the over night culture to an OD₆₀₀ = 0.1

-          Harvest at OD₆₀₀ = 1 (takes about 4 hours)
(centrifuge for 10 min at 5000 rpm)

-          Keep the supernatant (contains c-di-AMP)

-          1 x 250 ml
cook for 30 min on Bunsen burner in falcon tubes, centrifuge,  subsequent sterile filtration

-          1 x 250 ml
sterile filtration

-          Adding of 25 ml supernatant and 25 ml 2 x LB into a 50 ml Falcon
(4 Falcons for the cooked and filtrated medium; 4 Falcons for the filtrated medium)

-          Start by preparing each Falcon with 25 ml of the appropriate supernatant and the appropriate antibiotics
(in this case we need E/L, tet and cat; keep in mind that you need to supply your supernatant with 2 x antibiotic, because it gets diluted with LB later)

-          Now add 25 ml 2 x LB to each falcon. Mix the complete medium quickly and poor the plates!
(50 ml medium is needed to create 2 Agar Plates)

-          There should be 6 plates containing the cooked and filtrated supernatant and 6 plates containing only the filtrated supernatant (and 2 extra plates each)

-          Furthermore, prepare 2 plates, which contain no supernatant.
these plates will be needed for the pos. and neg. control

Transformation of GP 991 with chrom. DNA (cdaA::cat)

-          Inoculation of MNGE Medium (+ antibiotics) with the over night culture of GP 991 to an OD₆₀₀ = 0.1

-          Proceeded according to protocol of the AG Stülke

o   Pos. control: another cat resistance cassette (BP121)

o   Neg. control: H₂O

o   Actual experiment: GP 991 + GP 997

§  500 ng/μl used

-          plated as shown below:

 

Gel electrophoresis of digested cdaA (L. monocytogenes) and DAC domain (L. monocytogenes) (from 28.05.13)

Gel: Marker (100 bp) | undigested cdaA | double digested cdaA | undigested DAC domain | digested DAC domain | Marker (1 kb ladder) The bands of the digested sequences seem to have the expected sizes as there is no difference between the digested and the undigested DNA.

Ligation of cdaA (L. monocytogenes) and DAC domain (L. monocytogenes) into pGP172

Components	pGP172 + cdaA	pGP172 + DAC	Control
pGP172	1.7	1.7	1.7
cdaA	1.1	-	-
DAC domain	-	1.1	-
T4 liase buffer	1	1	1
T4 ligase	1	1	1
HPLC H2O	5.2	5.2	6.3
Total	10	10	10

 Ligation was performed overnight at 16.

 

Antibiotics

Antibiotic	Concentration	solve in:
Kanamycin	10 μg/ml	H2O
Lincomycin	25 μg/ml	H2O
Spectinomycin	150 μg/ml	H2O
Erythromycin	2 μg/ml	70% EtOH
Tetracyclin needs to be stored in the dark	12.5 μg/ml	70% EtOH
Chloramphenicol	5 μg/ml	70% EtOH

Media

1 x CSE - 0.5% Glc
20 ml	5 x C-Salts
1 ml	Tryptophan (5 mg/ml)
1 ml	CAF (2.2 mg/ml)
1 ml	III'-Salts
2 ml	Kaliumglutamat (40%)
2 ml	Nattriumsuccinat (30%)
1 ml	Glucose (50%)
ad 100 ml	dH2O

1 x LB
10 g	Trypton
5 g	Yeastextract
10 g	NaCl
1,5 %	Agar
ad 1000 ml	H2O

 

 

 

Preparation of cultures and medium

-          Bacillus subtilis wt 168 inoculation in 4 ml LB over day (28 °C and 200 rpm)

-          Over day 168 LB culture transferred to 2 x 50 ml CSE – 0.5% Glc medium
(50 μl of over day culture used; 30 °C and 200 rpm over night)

-          This strain is used for the production of c-di-AMP

-          Prepare 2 x 250 ml CSE – Glc medium and store these over night at 37 °C

-          Inoculation of GP 991 in 4 ml LB – L/B, tet

o   This strain is used for transformation

 

PCR for amplification of cdaA full length proteind and DAC domain from L. monocytogenes and Gel Extraction and purification of PCR products

PCR of full length protein:                           PCR of DAC domain:

Components	Volume (μl)	Components	Volume (μl)
forward primer (iGEM 8) [5 μM]	4	forward primer (iGEM 8) [5 μM]	4
reverse primer (iGEM 9) [5 μM]	4	reverse primer (iGEM 9) [5 μM]	4
DNTPs [12.5 mM]	2	DNTPs [12.5 mM]	2
DNA	2	DNA	2
5x HF buffer	10	5x HF buffer	10
HPLC water	27	HPLC water	27
Phusion polymerase	1	Phusion polymerase	1
Total	50	Total	50

4 μl of each primer were used instead of 2 μl to avoid the high selfannealing

rate!

Melting temperature: 60°C

Annealing Temperature: 54°C

Extension time: 2:30 min

The obtained PCR products were analyszed on 1% agarose gel.

Gel:

Marker (100 bp ladder) | full length cdaA | full length cdaA | DAC domain |

DAC domain | supposed control

Expected size for the PCR product of the full length protein: ~ 900 bp Expected size for the PCR product of the DAC domain: ~ 600 bp The supposed control should only contain the primers, but as a band could be detected, it has to contain as well genomic DNA from L. monocytogenes! → The gel pieces for the bands of the full length protein and the DAC domain were excised and the PCR products extracted and purified by adding the 3x volume of QG buffer and following the Quiagen PCR purification kit according to the manufacturer's protocoll. → The concentration was measured by NanoDrop: cdaA (L. monocytogenes): 81.0 ng/μl DAC domain (L. monocytogenes): 111.4 ng/μl

 

Digestion of cdaA from L. monocytogenes and DAC domain from L .monocytogenes and preparation for sequencing

Components	Volume (μl)
1500 ng insert	18.5 /13.5
SacI	4
BamHI	4
10x Fast Digest Buuffer	4
HPLC water	9.5/14/5
Total	40

Incubation for 30 min at 37°C

Preparation for Sequencing: Volume (μl)

Components	Sau_iGEM4	Sau_iGEM5	Spneu_iGEM 6	Spneu_iGEM 7
DNA	10	10	10	10
Forward primer	4	-	4	-
Reverse primer	-	4	-	4
Total	14	14	14	14

Sent for Sequencing!

→ sequencing was successful!

 

Restriction digestion of pGP172 with BamHI to check BamHI activity

Gel extraction and purification of pGP172 digested with BamHI and SacI

concentration of pGP172: 26.6 ng/μl

Components	Volume (μl)
Plasmid	20
BamHI	4
Fast Digest ? buffer 4	4
HPLC water	12
Total	40

The obtained PCR products were analyszed on 1% agarose gel.

Gel:

Marker (100 bp ladder) | undigested plasmid | BamHI digested plasmid | BamHI + SacI digested plasmid

 

For the undigested plasmid could be several bands detetced that represent the different states of the plasmid: linear, circular and super coiled. For the only BamHI digested plamid could be several bands but one main band be detected and for the double digested plasmid could be one band detecetd.

→ BamHI shows high activity, therefore the samples should be digested for 30 min

→ If you have a circular plasmid and need a BamHI and a SacI digestion, you should start with SacI to get a linear plasmid, then heat inactivate the enzyme and continue then with the BamHI digestion. This order is very important, because BamHI tends to cut non-specific regions in circular plasmids.

→ The gel piece for the band of the double digestes pGP172 was excised and the plasmid extracted and purified by adding the 3x volume of QG buffer and following the Quiagen PCR purification kit according to the manufacturer's protocoll.

→ The concentration was measured by NanoDrop: 17.8 ng/μl

 

PCR for amplification of cdaA from S. aureus and S. pneumoniae

S. aureus:

Components	Volume (μl)	Melting temperature: 58°C Annealing Temperature: 52°C Extension time: 2:30 min Expected product size: ~ 1.3 kb
forward primer (iGEM 4) [5 μM]	2
reverse primer (iGEM5)[5 μM]	2
DNTPs [12.5 mM]	2
DNA	2
5x HF buffer	10
HPLC water	31
Phusion polymerase	1
Total	50

S. pneumoniae:

Components	Volume (μl)	Melting temperature: 58°C Annealing Temperature: 52°C Extension time: 2:30 min Expected product size: ~ 1.3 kb
forward primer (iGEM 6) [5 μM]	2
reverse primer (iGEM 7) [5 μM]	2
DNTPs [12.5 mM]	2
DNA	2
5x HF buffer	10
HPLC water	31
Phusion polymerase	1
Total	50

 

Expected product size: ~ 1.3 kb

The obtained PCR products were analyszed on 1% agarose gel.

Gel: PCR product S.a. | PCR product S.a. | PCR product S.p. | PCR product S.p. | control (both primers only) | Marker (1 kb ladder)

Expected sizes for cdA of S. aureus und cdaA of S. pneumoniae: ~ 1.3 kb → For both PCR products the expected bands of about 1.3 kb could be observedand could be used for further experiments. → The gel pieces for the bands of the cdaA of S. aureus and S.pneumoniae were excised and the PCR product extracted and purified by adding the 3x volume of QG buffer and following the Quiagen PCR purification kit according to the manufacturer's protocoll. → The concentration was measured by NanoDrop: cdaA (S. aureus): 138.4 ng/μl cdaA (S. pneumoniae): 138.0 ng/μl