Observation of Back-up plates

-          Part 1, C1 and C2 probably very light pink

-          Part 2, C2 and C3 became light pink, but C1 stayed white (compare with restriction analysis 13.6.13)

 

Preparation of media for inoculation on Sunday (by Katrin Gunka)

-          For Plate Reader assay: 4 ml LB + 4 μl Cm or Amp for C1, C2 and C3 of parts 1 – 4 and part 6, negative control DH5α in 4 ml LB

-          For Plasmid Mini-Preparation: 10 ml LB + 10 μl Amp for inoculation of C2 Part 2

Test restriction of plasmids containing parts 1 – 8

-          Reactions/Master Mixes: see Excel-sheet “dropbox > iGEM > Reporter-Team > digestion system 2hour”

-          Plasmids: parts 1 – 6 (plasmids purified on 7.6.13) and part 7, 8 (plasmids purified on 11.6.13)

-          1x reaction with single enzyme:

Component	Volume
Enzyme	0.5 µl
Buffer 10x	1 µl
Plasmid DNA	200 ng
Total volume	10 µl

è     For double digestion:

           0.5 µl of each enzyme in case of EcoRI and PstI;

          for SpeI and PstI, ratio 1:2 is recommended à 0.5 µl SpeI and 1 µl PstI used

-          Incubation at 37°C for1.5 hours

-          Additon of 5xDNA-Loading Dye to whole reaction

-          Loading of 8 µl on agarose gel (~1.5 %, 1xTAE), QuickLoad 1 kb ladder as marker

-          Run at 200 V for 1.5 h in 1xTAE

-          EtBr staining ca. 45 min, destaining in water ca. 30 min

-          UV detection

 

Expected Fragments:

Part no.	Fragments for PstI and SpeI (bp)	Plasmid size (linearization with PstI or SpeI)(bp)
1	~900 + 2000	2948
2
3
4
Part no.	Fragments for PstI and EcoRI(bp)	Plasmid size (linearization with PstI or EcoRI)(bp)
5	??? (if RFP gene à same pattern as expected for parts 1 – 4; if no RPF gene à same pattern as expected for part 8)
6	917 2029	2946
7	140 2050	2199
8	53 2038	2091

 

Gel: See ppt-file “dropbox > iGEM > Reporter-Team > Geldoc“

Conclusions:

è     Partial digestion (next time: longer incubation time, less plasmid…)

è     For parts 1, 3, 4, 6, 7 and 8, the expected bands were observed (ok)

è     Band pattern of part 5 resembles that expected for parts 1 – 4 à this plasmid contains RFP gene

è     In case of part 2, SpeI was unable to cut the plasmid à no cleavage for SpeI single digest + linearization for SpeI/PstI double digest à plasmid contains probably something else (SpeI/XbaI scar at actual SpeI site?)

è     For cloning: terminator (part 7) and strong RBS (part 8) might be difficult to extract from the gel, since they are very short à think of other cloning strategy without gel extraction (e.g. “play” with resistances of plasmid backbones to digest backbone after cutting out part…)

 

Preparation of DarR PCR samples for sequencing

-          4 µl of DarR seq PCR product of reaction 1 + 1 µl of iGEM_34 (fwd) or iGEM_35 (rev)

Tubes:

kgun_1 à iGEM_34

kgun_2 à iGEM_35

-          Sequencing at G2L

SDS-PAGE of overexpressing cells (#811, 814, 1013) containing pGP172 + cdaA (from #81) and pGP172 + DAC (from #101)

 - take pelletized cells - add 5 μl of 5x SDS loading dye

 - add 10 μl ofZAP buffer - incubate for 10 min at 96°C

 → load samples on SDS PAGE and run gels at first at 80V, then turn to 100V

 - after running: cover gels with Coomassie Brilliant Blue staining solution and incubae for 30 min

 - wash the gels afterwards twiche with deionized H2O

 - destain gels overnight in 10% acetic acid with agitation

Gel:

first pre-experiment Marker (10 - 170 kDa ladder) | control (0 h) | control (1 h) | control (2 h) | 811 (0 h) | 811 (1 h) | 811 (2 h) | 814 (0 h) | 814 (1 h) | 814 (2 h)

Gel: first pre-experiment 1013 (0 h) | 1013 (1 h) | Marker (10 – 170 kDa ladder) | 1013 (2 h) | 1013 (4.5 h)

The T7 polymerase has a molecular weight of approximately 99 kDa. The full length cdaA protein from L. monocytogenes has a molecular weight of approximately 30.5 kDa, fused to a Strep-tag it has 31.5 kDa. The DAC domain of L. monocytogenes has a molecular weight of approximately 18.8 kDa, fused to a Strep-tag it has 19.8 kDa.

For the first 2 h after gene induction by IPTG no gene expression could be detected!

 

Gel: repeat pre-experiment control (0 h) | control (1 h) | 811 (0 h) | 811 (14 h) | 811 (2 h) | 814 (0 h) | 814 (14 h) | 1013 ( 0 h) | 1013 (14 h) | Marker (10 – 170 kDa ladder)

After 14 h could be gene expression for both T7 polymerase and DAC domain detected. For cdaA gene expression no signal could be detected. Therefore in further experiments cells should be incubated at least for 14 h to obtain high expression levels!

 

Continue: Repeat: Pre-experiment: Overexpression of cdaA and DAC domain by IPTG induction -measure OD600 of induced cultures

Culture	OD600	Saved cells [μl] at t0
#811	0.69	145
#814	0.79	127
#1013	1.82	55
control	2.20	46

- pelletize cells and store at - 20?C

 

Cryo-Stocks of E. coli transfomants (8, 7 C1)

Stored in -70 °C (red box)

 

Plasmid Mini-Preparation of parts 8, 7 C1

-          harvesting of ca. 5 ml pellets in 2 ml Epis for future Plasmid Mini-Preparation à stored at – 20 °C in red box

-          harvesting of remaining culture for today’s prep

è     Performed as on 7.6.13

NanoDrop – Plasmid concentrations

Part no.	c(DNA) [ng/µl]	A260/A280	A260/A230
7	67.0	1.98	2.25
8	62.7	1.89	2.08

 

DarR seq PCR purification

-          for DarR seq PCR reactions 1 - 4

-          with QIAquick PCR purification Kit (Qiagen), according to quick start manual

-          ca. 50 µl of PCR reaction + 250 µl of PB buffer

reactions 2 and 4 turned violet à addition of 10 µl NaAc 3.3 M, pH = 5.0

-          elution: 30 µl pre-warmed HPLC water directly applied to membrane, incubation for 2 min at RT, centrifugation

-          purified PCR products stored at – 20 °C in red box

Preparation of c-di-AMP supernatant without AB

·         168 innoculation of 1L CSE to an OD₆₀₀ = 0.1 at 9.00h (used preculture that grew over night [50 ml CSE at ])
11.00h: 0.384
12.00h: 0.69
12.20h: 0.82
12.50h: 1.035

·         centrifuge (5000 rpm, 10 min), cook for 30 min (preheat in microwave), centrifuge (5000 rpm, 5 min), sterile filtration

·         store in fridge

Microarray in Groningen

Repeat: Pre-experiment: Overexpression of cdaA and DAC domain by IPTG induction

- take over night cultures of #811, 814, 1011, 1012 and 1013 and measure OD600:

-inoculate afterwards 4 ml liquid media to an OD600 = 0.1

- add IPTG [1mM] to a final concentration of 1 μM to the liquid cultures

- incubate at 16°C for 12 – 14 h

 

Colonies on the plates: parts 8, 7 C1

10 ml LB medium with 10 µl antibiotics, overnight culture for mini-prep

backup plates: C1, C2, C3 for part 8

 

Colonies on the plates: parts 8, 7 C1

Preparation of 250 ml LB broth media (stored on shelf above bench)

10 ml LB medium with 10 µl antibiotics, overnight culture for mini-prep

backup plates: C1, C2, C3 for part 8

Re-streak of E.coli clones from prtas 6 and 7 on new LB^Chloram plates

Preparation of new LB^Chloram plates (500 ml) and Preparation of 250 ml LB broth media (stored on shelf above bench)

 

PCR with DarR primers with different enzymes and different buffers

-          dilution of primer stocks 1:20 (100 pmol à 5 pmol):

95 µl HPLC water + 5 µl primer 100 pmol

-          preparation of dNTP mix à dilute stocks 1:8 (100 mM à 12.5 mM):

50 µl of dATP 100 mM

+ 50 µl of dGTP 100 mM

+ 50 µl of dTTP 100 mM

+ 50 µl of dCTP 100 mM

+ 200 µl dH₂O

è     diluted primers and dNTP mix stored in red box at -20 °C

 

1x reaction (50 µl)

Component	Volume
Buffer (5x GC buffer or 5x HF buffer)	10 µl
dNTP mix (12.5 mM each)	2 µl
Primer fwd iGEM_34 (5 pmol)	2 µl
Primer rev iGEM_35 (5 pmol)	2 µl
Chromosomal DNA M. smegmatis	2 µl
DNA-Polymerase (Phusion or PhuS) or dH2O for water control	1 µl
dH2O	31 µl

 

-          preparation of master mix for 6 reactions containing

Component	Volume
dNTP mix (12.5 mM each)	12 µl
Primer fwd iGEM_34 (5 pmol)	12 µl
Primer rev iGEM_35 (5 pmol)	12 µl
dH2O	186 µl

-          addition of template (chrom. DNA/dH₂O), polymerase and buffer individually, then addition of 37 µl master mix

è     For tested combinations of buffer and Pol: see table for gel loading

 

-          PCR protocol (cycler 7; folder “Katrin” > “iGEM” > “DarR seq”)

Step	Temperature	Time
Initial denaturation	98.5 °C	5 min
Denaturation	98.5 °C	30 s
Annealing	60 °C (TA = TM (≈66 °C) – 6 °C)	35 s
Elongation	72 °C	2 min (Phu  needs more time than Phusion!)
Final elongation	72 °C	10 min
Hold	15 °C	∞

-          1 % Agarose-1xTAE gel

-          4 µl PCR reaction + 1 µl 5x DNA Loading Dye

-          3 µl 1 kb ladder (Quick Load)

-          Run at 100 V in 1xTAE buffer

-          Staining in EtBr and destaining in water

-          UV detection

M	1	2	3	4	5	M
1 kb ladder (QuickLoad)	HF	GC	HF	GC	Water controlHF	1 kb ladder (QuickLoad)
	Phusion		PhuS

è     Reactions stored at - 20°C in 50 ml Falcon

Pre-experiment: Overexpression of cdaA and DAC domain by IPTG induction

- take over night cultures of #811, 814, 1011, 1012 and 1013 and measure OD600:

-inoculate afterwards 4 ml liquid media to an OD600 = 0.1

Culture	OD600	Used μl for inoculation
#811	0.67	600
#814	1.39	290
#1011	1.29	310
#1012	1.18	340
#1013	0.95	420
control	1.54	220

- add IPTG [1mM] to a final concentration of 1 μM to the liquid cultures for

induction - incubate at 16°C for 0 – 2 h - measure OD600 of induced cultures → take equivalent amount of cells after 0 h, 1 h and 2 h: 100 [μl] /OD600

Culture	OD600	Saved cells [μl] at t0
#811	0.60	167
#814	0.64	167
#1013	0.87	115

-pelletize cells and store at -20?C