Team:Goettingen/NoteBook w1

Cryo-Stocks of E. coli transformants (C1, parts 1 – 7):

·         900 μl E.coli ON culture + 100 μl DMSO 100% in special tubes (ask Katrin or Katrin^^)

·         vortex

·         store at – 70 °C (red box)

Backup plates:

Storage at 4 °C

 

Plasmid Mini-Preparation of parts 1 - 7:

ON cultures of C1 for parts 1 – 7 with kit “NucleoSpin? Plasmid” von Macherery-Nagel according to manual pp. 16-17 (NucleoSpin? Plasmid protocol for purification of high copy plasmids)

Step 5: recommended washing of silica membrane with buffer AW was performed

Step 7: Elution with buffer AE pre-heated to 50 – 60 °C (in future: DON’T elute with this buffer, use pre-heated HPLC-H₂O instead! No one knows what’s inside the buffer and its components (EDTA?) could interfere with sequencing and other reactions)

 

NanoDrop – Plasmid concentrations

Part no.	c(DNA) [ng/μl]	A260/A280	A260/A230
1	84.6	1.94	2.18
2	79.8	1.94	2.10
3	151.0	1.89	2.24
4	29.5	1.91	2.07
5	154.9	1.88	2.25
6	88.9	1.92	2.08
7	5.9	14.08	1.87

Stored in red box at - 20°C

Primers arrived!

iGEM_32 ~ 37: dissolved in HPLC water, stored at -20^oC in our box

100uM stock , for PCR dilute 1:20 in HPLC water.

Primer 32 and 33 are strong in forming 2^nd structures, increase the amount in PCR

Preparation of cryo-cultures of #811, 814, 1011, 1012 and 1013

preparation of cryo-cultures:

- take 900 μl of overnight culture

- add 100 μl 100% DMSO (filter sterile)

→ store at - 80°C

 

Media preparation:

1000ml LB+Ampicillin Agar => ca. 50 Plates (Black Code)

Transformation:

Number	whereabout
B0034	P5 2 M

Colonies on the plate: parts 1- 7:

4ml LB with antibiotics, overnight culture for mini-prep[C1]

Backup plates: C1,C2,C3 for each part

Continue of transformation of competent E. coli cells with pGP172 + cdaA (from #81) and pGP172 + DAC (from #101)

only clones #811 and #814 (expressing pGP172 + cdaA) grew overnight

→ inoculate them in liquid LB media

→ grow normally in LB media clones # 1011, 1012, 1013 were directly inoculated in liquid LB medium

 

Preparation of Antibiotic Stocks

1000x Ampicillin 10 1mL Stocks (Freezer red box)

1000x Chloramphenicol 10mL Stock (Falcon in Freezer)

Media preparation:

 250ml *4 Chl

 [use the ones marked with “5^th..6.13 LB^chl” on EVERY plate first. ]*

 250ml *1 Amp  (marked with black)

primer design<ordered>

iGEM_36	DarR operator sequence + prefix
iGEM_37	DarR operator sequence + suffix

Transformation:

Number	Mark
J23117	1
J23116	2
J23110	3
J23118	4
J61101	5
BBa_BE0240-Chl	6 ---Chl
BBa_B0015	7----Chl
B0034	8

Resuspended DNA from iGEM kit (already stored in red box in -20 frigde):

Number	whereabout
BBa_E0204-Amp	P5 12 M
BBa_QO3121	P5 20 N

 

 

Transformation of GP 911

·         According to the protocoll of AG Stülke

o   Started at 8.00; OD₆₀₀ = 1.4 at 11.00h

o   For the expression mix, the CSE supernatant was used instead of water.

Results:

	controls on LB medium, containig the appropriate AB. left: neg. control: 991 + water right: pos. Control: 991 + BP121 (another cat cassette)
	the pos. control on LB medium with sterile filtrated supernatant
	the pos. control on LB medium with cooked and sterile filtrated supernatant

 

·         No growth of the actual experiment on these plates (GP 991 + GP 997)

·         This might be due to the fact that the expression mix, used during the trafo, contained the supernatant containing all three antibiotics (E/L, tet, cat)

o   Redo this next week with supernatant lacking the AB’s

Continue of transformation of competent E. coli cells with pGP172 + cdaA (from #81) and pGP172 + DAC (from #101)

E. coli cells expressing pGP172 + cdaA (from #81) are growing very slowly and have to be incubated longer

→ chose four clones and name them: #811, 812, 813 and 814

→ re-streak the om LB Amp100 plates

E. coli cells expressing pGP172 + DAC (from #101) are growing normally

→ chose three clones and name them: #1011, 1012 and 1013

 

primer design<ordered>, found the correct sequence for DarR

iGEM_32	Primer DarR + Prefix forw.
iGEM_33	Primer DarR + Suffix rev.
iGEM_34	Primer DarR sequencing forw.
iGEM_35	Primer DarR sequencing rev.

Creation of c-di-AMP supernatant, which is used for the plates

 

·         - growth curve of 168 in CSE:
9.00h:     0,1
11.00h:   0.37/0.384
12.00h:   0.72/0.71
12.30h:   0.99/0.95

·         continued as described on 29.05.13

o   Addition: the prepared falcons containing the 25 ml supernatant were preheated in the 37 °C room. That’s makes pouring the plates easier

 

Sequencing of plasmids from clone #81 and #101

Component	81F	81R	101f	101R
DNA	10	10	10	10
Forward primer	4	-	4	-
Reverse primer	-	4	-	4
Total	14	14	14	14

 Sent for sequencing!

 

Transformation of competent E. coli cells with pGP172 + cdaA (from #81) and pGP172 + DAC (from #101)

Transform competent E. coli cells according to the protocoll mentioned above (30.05.2913) with pGP172 + cdaA or pGP172 + DAC, respectively.

For the transformation were 5 μl of plasmid DNA used. The plates were incubated overnight at 37°C.

 

Gel electrophoresis of colony PCR from 31.05.2013

The obtained PCR products were analyszed on 1% agarose gel. Clones #81 – #90 should contain pGP172 + cdaA (L. monocytogenes). Clones #101 – #110 should obtain pGP172 + DAC domain (L. monocytogenes). Therefore a band of about 900 bp were expected for clones #81 – #90 and a band of about 600 bp for clones #101 - #110

Gel: Marker (100 bp ladder) | clones #81 – #90 | clones #101 – #102 | Marker (1kb ladder)

Gel: Marker (100 bp ladder) | clones #103 – #110 | control (w/o insert)

For all clones could be a specific band and therefore also a specific PCR product were obtained. The plasmids from clone #81 and #101 were extracted and purified by peqlab Miniprep kit according to the manufacturer's protocoll.

→ The concentration was measured by NanoDrop: 81/cdaA (L. monocytogenes): 27 ng/μl 101/DAC domain (L. monocytogenes): 27 ng/μl

 

Digestion of pGP172 + cdaA (L. monocytogenes) and pGP172 + DAC domain (L. monocytogenes) with SacI and BamHI

Components	pGP172 + cdaA	pGP172 + DAC domain
Plasmid (300 ng)	11.1	11.1
SacI	1	1
BamHI	1	1
Fast Digest Buffer	1	1
HPLC H2O	5.9	5.9
Total	20	20

The digestion products were analyzed on 1% agarose gel.

Gel: Marker (100 bp) | undigested pGP172 | cdaA insert | DAC insert | ligation control | digested pGP172 + cdA | digested pGP172 + DAC

The digested plasmids show bands for the undigested pGP172, for the corresponding insert as well as a band for the undigested pGP172 including the insert. Furthermore could be an additional band in the ligation control as well as in the digested pGP172 + cdA and the digested pGP172 + DAC observed. This could be due to unspecific digestion or the digestion by only one enzyme. Therefore the cloning seems to be successful and should be further verified by sequencing!