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InterLab Study

our construct	BBa	promotor strength
pILS 1	J23117	162
pILS 2	J23106	1185
pILS 3	J23101	1791
positive	J23151 1bp mutant from J23114	256 (J23114)

12.08.2015

TOP10 pILS2 and pILS3 10°C

• because not all the colonies grew properly the experiment was repeated for TOP10 pILS2 and pILS3 at 10°C

11.08.2015

• the protocol on the iGEM website was followed:
• The samples were diluted to OD₆₀₀ = 0.5:
  • From all 60 samples the OD₆₀₀ was measured using a nanoDrop
  • samples were diluted to achieve a final OD₆₀₀ of 0.5
  • dilution was checked by again measuring the optical density of the samples
  • in case of a value outside 0.5 +- 5% the diluation was repeated based on the new values
• fluorescence was measured using a platereader an an excitation of 485nm with a bandpassfilter of 20nm and emission at 530nm with the filter width.
  • all fluorescence data was measured three times in three 96-well plates (technical replicates)

10.08.2015

• AS the BL21 strain carries the same resistance (Cml) as the plasmid does and it wasnot possible to sort out transformed colonies by using the fluorescence as guide we decided not to use BL21 in the study
• from each plate two test tubes with 5 mL LB medium (+Cml) were inoculated
• grown them for 8 h on 37°C, 220 rpm
• after 8 h transfered half of the test tubes to 10°C

09.08.2015

• For the other 2 strains: Three colonies of each strain were picked and stricken out on plate
• incubation o/n @ 37°C

08.08.2015

Used E.coli strains are TOP10 (cloning strain) and Bl21pLysS + Arctic Express (expression strain) Each construct was transformed into the different strain. Constructs:

• Interlab positiv
• Interlab negativ
• pILS 1 (2)
• pILS 2 (2)
• pILS 3 (1)

The antibiotic resistance of the plasmid is chloramphenicol. The expression strain Bl21pLysS has the same resistance. For o/n cultures only the visual positiv colonies (expression of gfp → greenish colonies will be choosen. Due to the fact of having no resistance (TOP10) and another antibiotic resistance (gentamycin) for Arctic Expression the selection by these strain is easier.

• transformation was performed according to the protocol Transformation

2015/08/05

Inoculation of 5 ml liquid culture (RE)

• pILS_1 (LB_cml)
• pILS_2 (LB_cml)
• pILS_3 (LB_cml)

2015/08/04

Transformation (RE)

• pILS_1
• pILS_2
• pILS_3

2015/08/03

Test digest: Interlab Study (LS)

• Interlab Study: pILS1, 2 and 3–>EcoRI HF and SpeI HF

6xMasterMix
6µl	EcoRI HF
6µl	SpeI Hf
12µl	Cut smart
66µl	dH2O

2015/08/02

Picked colonies from ligation plate (interlab study) (?)

Miniprep: pILS1,2,3 (JN)

• qiagen kit
• eluted in 30µl dH2O

plasmid	concentration
pILS1 (1)	84,4
pILS1 (2)	51,7
pILS2 (1)	60,7
pILS2 (2)	107,3
pILS3 (1)	11,2
pILS3 (2)	5,1

2015/08/01

Digest of the ILS plasmids (RE)

• Bba_K823013 with SpeI and PstI-HF
• Bba_K823008 with SpeI and PstI-HF
• Bba_K823005 with SpeI and PstI-HF
• Bba_I13504 with XbaI and PstI-HF

• 1 µl of each restr. enzyme
• 5 µl CutSmart
• 10 µl DNA
• 33 µl dH2O
• incubated for 1 h at 37°C

analysis on 1 % agarose gel:

---

Ligation (RE)

• InterLab study:

Backbone	Bba_K823013	Bba_K823008	Bba_K82_3005
Backbone amount [µl]	2.17	1	2.08
GFP (Bba_I13504) amount [µl]	3.09	3.09	3.09

Transformation of the ligation products (5 µl) into E.coli TOP10 (RE)

• pILS1-3 were plated on LB-cml

26.07.2015

• from the first miniprep another try for GFP (miniprep with 105.1 ng/µL) digest was done

	volume [µL]
DNA	10
10x cutSmart	5 µL
Xba1	1 µL
Pst1-HF	1µL
water	33 µL

• NEB Cloner proposed 5-15 min @ 37°C but digest over night should also be possible
• digest was carried out for 2.5h @ 37 in Thermocycler
• sample was loaded on a free lane of a before used 1% agarose gel
  • 5µL 1kb ladder was used

Results

Lane of digested GFP.

• Only one band is visible so the digest was not succesfull
• Additionally the 1kb-ladder was not visible on the gel.

Before further experiments are carried out, the DNA from the second miniprep has to be sequenced to prevent the right digest of the wrong DNA!

25.07.2015

• A miniprep for all 4 InterLabStudy-constructs was carried out

construct	yield [ng/µL]
BBa_I13504	111.9
BBa_K823005	176.6
BBa_K823008	76.3
BBa_K8230	109.0

• From each o/n culture a plate culture was stroken out to have cells for further minipreps

24.07.2015

• from all four constructs colonies still stored @ 4°C cells were taken and put into 3 mL LB-medium + Cml
• cells were grown o/n for miniprep the next day

23.07.2015

• samples were digested with the right volumes

	K823005	K823008	K823013-2	I13504-1
Plasmid	42 µL	30 µL	28 µL	30 µL
Pst1	1 µL	1 µL	1 µL	1 µL
Spe1	1 µL	1 µL	1 µL
Xba1				1 µL
5x buffer	5 µL	5 µL	5 µL	5 µL
aqua dest	1 µL	13 µL	15 µL	13 µL

• 1% agarose gel was loaded and run 45 min @ 110V

Digest of the samples with Pst1 and Spe1 (for the backbones) and Xba1 (for the insert) respectively. Sample loading should have been all three backbones and then the insert, but samples seem to be swapped.

Results

• samples seem to be swapped
  • The GFP insert was assigned to the first construct with about kbp length → There the digest may not have worked and therfore only one band is visible
  • The other bands were cut out and marked as Insert 1 to 3 because assignment to the different samples was not clear.
  • It is additionally unclear, if there has been succesfull digestion because the loss of 35 bp can't be seen on the gel
• The GFP-insert digest will be done again
• Backbone samples were stored @ 4°C

22.07.2015

• samples were loaded on a 1% agarose gel and run @ 110V for 30 min
• there were no bands visible

digest of the InterLab constructs failed.

21.07.2015

• constructs were diluted to reach final concentration of 1µg/µL

starting construct	dilution
Bba_K823005 46.4 µg/µL	45.4 µL aqua dest + 1µL
Bba_K823008 67.9 µg/µL	66.9 µL aqua dest + 1µL
Bba_K823013-2 72.7 µg/µL	71.7 µL aqua dest + 1µL
Bba_I13504-1 66.8 µg/µL	65.8 µL aqua dest + 1µL

• digestion was carried out for 1.5 h @ 37°C following the scheme and the protocol

	K823005	K823008	K823013	I13504
Plasmid	3 µL	3 µL	3 µL	3 µL
PstI	1µL	1µL	1µL	1µL
SpeI	1µL	1µL	1µL
XbaI				1µL
5x buffer	5µL	5µL	5µL	5µL
aqua dest	40µL	40µL	40µL	40µL

• after digestion enzymes were inactivated by 2min 80°C treatment
• samples were stored @ 4°C