Team:Goettingen/NoteBook w0
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Revision as of 20:26, 30 September 2013
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Selection of clones from transformation of pGP172 + cdaA (L. monocytogenes) and pGP172 + DAC domain (L.monocytogenes)
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 |
![](https://static.igem.org/mediawiki/2013/b/b7/Goe-timeline-dot.png)
Transformation of pGP172 + cdaA (L.monocytogenes) and pGP172 + DAC domain (L. monocytogenes)
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
![](https://static.igem.org/mediawiki/2013/b/b7/Goe-timeline-dot.png)
Prepare of agar plates.
Transformation of GP 991 with chrom. DNA. (cdaA::cat)
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 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.
![](https://static.igem.org/mediawiki/2013/b/b7/Goe-timeline-dot.png)
Antibiotics and media preperation.
Preparation of cultures and medium for c-di-AMP uptake experiment.
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 |
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
Digestion of cdaA from L. monocytogenes and DAC domain from L .monocytogenes and preparation for sequencing
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 irgendwas stimmt hier mit der ladder nicht?? 12 banden statt nur 10! Abstand zwischen den beiden PCR prdukten stimmt aber! Daher weiter verwendet! 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!
![](https://static.igem.org/mediawiki/2013/b/b7/Goe-timeline-dot.png)
Restriction digestion of pGP172 with BamHI to check
BamHI activity
Gel extraction and purification of pGP172 digested with
BamHI and SacI
PCR for amplification of cdaA from S. aureus and
S. pneumoniae
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
|