Transformation

Transformation of all red-light sensor parts separately. DH5alpha cells were transformed using electroporation (V=1600V) with following constructs:

Part alias : Part Name - Plasmid - Year of Distribution, Plate Number

iGEM R1 : R0082 pSB1A3 2010, P1

iGEM R2 : I732017 pSB1A2 2010, P2

iGEM R3 : I15010 pSB2K3 2010, P3

iGEM R4 : K098010 pSB4C5 2010, P3

Each electroporation was performed with 1,5µl bio-brick plasmid (each disolved in 10ul ddH2O before) and 40µl competent DH5alpha cells according to the protocol kindly provided by Andrea Mueckl

Afterwards cells were incubated in 1ml SOC medium for 1,5 h at 37°C, 200rpm and subsequently plated on antibiotic containing LB-agar plates (50µl, 100µl, 200µl) and incubated at 37°C over night and then kept at 4°C.

Transformation

Transformation of one red part sensor (iGEM R1) with slightly modified protocol: electroporation performed at 1500V with 1 µl Plasmid (part previously disolved in 10ul ddH2O)and 40ul competent DH5alpha. Otherwise same procedure as before.

MiniPrep

Testing clone: Mini-Prep using Zymoresearch DNA Kit, Digestion with EcoRI and PstI in NEB-Buffer 4, Gel (expected fragments 0,1 kbp (=R1) and 2,0 kbp (=pSB1A2))

Transformation

Transformation of

Part alias : Part Name - Plasmid - Year of Distribution, Plate Number

iGEM R2 : I732017 pSB1A2 2010, P2

iGEM R3 : I15010 pSB2K3 2010, P3

iGEM R4 : K098010 pSB4C5 2010, P3

using 1510V and 1ul DNA for transformation. DNA was just added to the cells and gently mixed by stirring it with the pipette tip. (Andrea Meyer's protocol) Of the 1ml SOC culture, 50ul were spread out directly on agar plates. Rest was spun down (2000rpm, 1min) and pellet was resuspended in 100-150ul. Subsequently, 50ul were spread out.

Transformation

Transformation of:

Part alias : Part Name - Plasmid - Year of Distribution, Plate Number

iGEM R3 : I15010 pSB2K3 2011, P3

iGEM tRNA : K228001, pSB1A2 2011, P4

iGEM Term : B0015 pSB1AK3 2011, P1

iGEM RBS : J44001 pSB1A2 2011, P1

iGEM T7 : I712074 pSB1AK8 2011, P1

since the transformation with iGEM R3 did not work, we increased the plasmid amount to 2 µl

afterwards the procedure was performed as mentioned above and the cells were incubated over night (15 h) at 37 °C and cooled until monday at 4 °C

Transformation

Transformation of R3 once more using BL21 and inducing pSB2K3 plasmid using 1mM IPTG in SOC-Medium

MiniPrep

Plasmid Isolation (MiniPrep) from all over night cultures

Digestion

- Restriction digestion with EcoRI and PstI in NEB-Buffer 4

- Gel run with digestion: 100 V, 1 h 10 Min, 1 % agarose

- CyberGold Staining: 2 µl on shaking plate for 30 mins @ RT

Transformation

Transformation of DH5alpha with:

iGEM R3 (see above) on A, C and K resistance plates

iGEM High Copy Plasmid, Plate 1 2011 1G, psB1A3 with insert: BBa_J04450, Resistance: A

iGEM Low Copy Plasmid, Plate 1 2011 3C, psB3C5 with insert: BBa_J04450, Resistance: C

plated on agar plates over night

Digestion

Gel run with digestion: 90 V, 1 h 30 Min, 1 % agarose

CyberGold Staining: 2 µl on shaking plate for 30 mins @ RT

Digestion

Gel run with newly digested and undigested plasmid as control: 100 V, 1 h 30 Min, 1 % agarose

Transformation

Transformation of R3 in E. coli D1210

Digestion

2 % Agarose with Ethidiumbromid, 90V, 1.5 h

Digestion of 2 µg Plasmid DNA with high fidelity 1.: EcoRI and PstI enzymes and 2.: just with PstI

Digestion

1.5 % Agarose with Sybr Gold (Dilution: 1 µl/10ml), 90V, 1.0 h

MiniPrep

MiniPrep of BBa_K238013 BBa_K228000 BBa_K322127

MiniPrep

MiniPrep of R3

Comparison of "new" (ordered) enzymes vs. "old" ones

Cloning of the red light sensor with parts R1,R2,R4 (see cloning draft)

ONC of part containing colonies in 5ml liquid culture

Ligation

Agarose gel of ligation products to evaluate ligation problems

MiniPrep

MiniPreps of Parts

-Cloning of the red light sensor with parts R1,R2,R4 (see cloning draft)

-ONC of part containing colonies and red light sensor construct colonies in 5ml liquid culture

-Cloning of the blue sensor (only step1) with parts K228000, RBS (see cloning draft)

-Cloning of the blue sensor (step2) with parts: Y44001 (RBS) + K22800 = A1 and B 0015 (Term)

-Cloning of the red light sensor with lacZ (R2)

Digestion

- gel run of all products (110 V, 1%, 1h): lanes: 2log ladder, RBS undigested (=ud), RBS digested (dig), K22800 ud, K22800 dig, A1 ud, A1 dig, Term ud, Term dig, L1 ud, L1 dig, L2 ud, L2 dig, R2 ud, R2 dig 6 µl each lane gel:

Transformation

-Transformation of both ligations (red and blue light) and incubation on plates over night:

- blue light on amp and kan

- red light I on amp plate not protected from light, lightning during electroporation

- red light II on amp 1 plate protected from light, 1 plate not protected from light

MiniPrep

- plasmid isolation of all red light sensor parts and the successfully transformed colonies from 21.06.11 concentrations in ng/µl

R1 93.5 R2 108.0 R3 37.0 R4 263.0 RBS 47.5 tRNA 154.0 Term 65.0 Ligation of transformed colony (21.6.11) I 95.9 Ligation of transformed colony (21.6.11) II 63.5 T7 106.0 high copy plasmid 142.0 low copy plasmid 66.5

Digestion (EcoRIxPstI) and ligation of K238013, tRNA in pSB3C5, blue light construct (j44001-K22800-B0015), Reporter construct (T7-R2)

Digestion (EcoRIxPstI) of K238013, tRNA in pSB3C5, blue light construct (j44001-K22800-B0015), Reporter construct (T7-R2)

Digestion

Digestion (ExP) of B0015, pSB1A3 (not needed), pSB3C5

Agarose Gel of K238013, tRNA in pSB3C5, blue light construct (j44001-K22800-B0015), Reporter construct (T7-R2)

Amplification

PCR to amplify certain parts out of the red light sensor of edinburgh: K322123, K322124, ompR:

PCR-Program:

1) Initial Denaturation: 96°C, 4'

2) Touchdown (8x)

   Denat.: 96°C, 30 s
   Annealing: 65°C -> 61,5°C; -0,5°C/cycle, 20 s
   Extension: 72°C, 4'

3) Constant Temp. (22x)

   Denat.: 96°C, 30 s
   Annealing: 61°C, 20 s
   Extension: 72°C, 4'

4) Final Extension: 72°C, 8'

5) Hold: 4°C, oo

PCR-mix (8x) (Taq-PCR-Kit; NEB):

water (autoclaved): 326 µl

standard buffer (10x): 40 µl

dNTPs (10mM): 8 µl

Taq-Pol: 2 µl

47 µl per reaction + 1 µl of each primer + 1 µl DNA -> 50 µl reaction volume

samples:

1) negative control: primer (10 µM) + water

2) DNA: 1,5 ng; primer (10 µM)

3) DNA: 15 ng; primer (10 µM)

4) DNA: 1,5 ng; primer (5 µM)

5) DNA: 15 ng; primer (5 µM)

6) DNA: 1,5 ng; primer (1 µM)

7) DNA: 15 ng; primer (1 µM)

Agarose

Agarose Gel of ladder, R2, T7, Pol, lac-Pol(R2-Pol) ligation, T7-R2 ligation

analytical agarose gelelectrophoresis of the PCR samples from 01-07-11: (1%, 1x TBE, 120 V, 400 mA, 90 min)

10 µl PCR sample + 2 µl loading buffer -> 10 µl per well

lanes:

1.) 2-log

2) negative control: primer (10 µM) + water

3) DNA: 1,5 ng; primer (10 µM)

4) DNA: 15 ng; primer (10 µM)

5) DNA: 1,5 ng; primer (5 µM)

6) DNA: 15 ng; primer (5 µM)

7) DNA: 1,5 ng; primer (1 µM)

8) DNA: 15 ng; primer (1 µM)

9) 2-log

Amplification

repetition of PCR to amplify certain parts out of the red light sensor of edinburgh: K322123, K322124, ompR:

Phusion High-Fidelity PCR-Kit (NEB/Finnzymes)

PCR-Program:

1) Initial Denaturation: 98°C, 4'

2) 30 cycles of:

   Denat.: 98°C, 30 s
   Annealing: 60°C, 15 s
   Extension: 72°C, 2´ 30 s

3) Final extension: 72°C, 8'

4) Hold: 4°C, oo

Samples

PCR-samples were prepared with HF- and GC-buffer, respectively. End volume of the reaction setups was 50 ul.

DMSO-concentration was 100%; primer concentration was 10 uM, which equals an end conc. of 500 nM (2.5ul) or 200 nM (1ul).

DNA-template [150 ng/ul] was diluted 1:100 to a conc. of 1.5 ng/ul -> 1 ul of dilution was used per reaction.

Samples were prepared as follows (same setup for HF- and GC-buffer):

Digestion

Digestion of J44001, K228000

Preparative gel run with the digested parts (1% agarose, 1xTAE, 120 V 400 mA, 1.5 h)

Isolation of the bands out of the gel

Preparation with a gel isolation kit (freeze ´n squeeze, bio-rad)

work-up of PCR-run (05-07-2011): gel analysis (1% agarose, 1xTBE, 120 V, 400 mA, 1.5 h) of HF-buffer-samples

gel analysis (1% agarose, 1xTBE, 120 V, 400 mA, 1.5 h) of GC-buffer-samples

isolation of right bands out of the GC-gel

DNA preparation with a gel isolation kit (freeze ´n squeeze, bio-rad)

restriction digest with EcoRI and SpeI

MiniPrep

mini-prep of culture (4 ml) from DH5alpha (05-07-2011) (qiagen qiaprep spin miniprep kit) -> 82 ng/µl in 50 µl elution buffer

restriction digest of 0.5 µg DNA with EcoRI and PstI

gel analysis of restriction digest (1% agarose, 1xTAE, 120 V, 400 mA, 1.5 h)

Ligation

Ligation (total volume: 50 µl; 1.5 µg of each DNA sample; 5 µl T4-ligase buffer (10x); 1 µl Quick-Ligase; add to 50 µl with nf H20; 60`@ 37°C)

Ligation of:

1. K228000 + RBS

2. K322127 + supD-tRNA

3. lacZ + T7-Promotor

preparative gel for ligation from 07-07-11; 1% TAE, 8 wells, 120 V, 400 mA, 90';

30 µl DNA + 6 µl loading dye (6x) -> 36 µl, 35 µl loaded on gel

7,5 µl 2-log DNA ladder

Ethanol precipitation (1/10 of volume 3M NaAc (pH 5.8), 2.5 X EtOH (100%, icecold)) of:

- Backbone (T7-Promotorpart, cut with S, P): 200 µl

- RBS (cut with S, P): 70 µl

- K228000 (cut with X, P): 90 µl

- K322127 (PCR-product redlight w/o lacZ; cut with E, S): 180 µl

- lacZ (cut with X, P): 90 µl

Add NaAc and EtOH (100%), invert 5x, incubate 30' @ -80°C

centrifuge 30' @ 4°C, 10000 rcf

no visible pellet -> centrifuge again, 30' @ 4°C, 18000 rcf

discard supernatant

wash with EtOH (70%, icecold) -> centrifuge 5' @ 4°C, 18000 rcf

discard supernatant

dry pellet (30' @ RT)

resuspend in 10 µl nuclease-free H20 (at no time visible pellet. nanodrop measurements suggested no detectable amount of DNA)

Restriction

Restriction digest (total volume: 50 µl; Enzymes: 1 µl each; 5 µl NEB 4 buffer (10x); incubation: 1 h @ 37°C; inactivation: 20 ' @ 80 °C) of PCR samples 2 and 4 (HF-buffer) from 05-07-11:

DNA: 10 µl (sample 2: 1 µl primer [10 µM], -DMSO; sample 4: 1 µl primer [10 µM], +DMSO)

H20: 33 µl

Enzymes: E, S

Preparative agarosegel (1%, 1xTAE, 8 wells, 120 V, 400 mA, 90'):

35 µl restriction digest + 7 µl loading dye (6x) -> 42 µl; 40 µl loaded on gel

7 µl 2-log DNA ladder

lanes:

1.) 2-log

2.) PCR-sample 2 from 05-07-11

3.) PCR-sample 4 from 05-07-11

Digest

Restriction digest (total volume: 50 µl; DNA: 1,5 µg; Enzyme: 1 µl each; 5 µl NEB 4 buffer (10x); incubation: 1 h @ 37°C; inactivation: 20 ' @ 80 °C) of the following parts:

- lacZ: 94 ng/µl -> 16 µl; H20: 27 µl; Enzymes: X, P

- T7-Promotor: 106,6 ng/µl -> 14 µl; H20: 29 µl; Enzymes: S, P

- SupD-tRNA: 150 ng/µl -> 10 µl; H20: 33 µl; Enzymes: E, X (#1, #2: double preparation for validation of different subsequent DNA purification steps)

- K238013 (blue light promotor): 233 ng/µl -> 6,5 µl; H20: 36,5 µl; Enzymes: E, X

- B0015 (Term.): 141 ng/µl -> 10,5 µl; H20: 32,5 µl; Enzymes: E, S

- RBS: 108 ng/µl -> 14 µl; H20: 29 µl; Enzymes: S, P

preparative agarosegel (1%, 1xTAE, 8 wells, 120 V, 400 mA, 90'):

35 µl restriction digest + 7 µl loading dye (6x) -> 42 µl; 40 µl loaded on gel

7 µl 2-log DNA ladder

1) DNA ladder

2) lacZ

3) T7 Promotor

4) supD tRNA

5) K328013

6) RBS

preparative agarosegel (2%, 1xTAE, 8 wells, 120 V, 400 mA, 90'):

35 µl restriction digest + 7 µl loading dye (6x) -> 42 µl; 40 µl loaded on gel

7 µl 2-log DNA ladder

0) DNA ladder (pipette tip fell off)

1) DNA ladder

2) B0015 (Term.)

Ligation

Ligation (total volume: 30 µl; 10 µl of each DNA sample; 3 µl T4-ligase buffer (10x); 1 µl Quick-Ligase; 6 µl nf H20; 30`@ 37°C)

Ligation of:

1. lacZ + T7-Promotor

2. K322127 (PCR-Product) + supD-tRNA

3. K238013 + B0015

MiniPrep

Preparation of 24 overnight-cultures (5 ml each) for miniprep

Picking

picking of 3 clones (labelled a, b and c) per positive (= visible colonies) plate:

plate 1: lacZ + T7-promotor AFTER purification -> no colonies visible; Kan-resistance

plate 2: lacZ + T7-promotor BEFORE purification -> colonies; Kan-resistance

plate 3: PCR-product (from K322127) + supD-tRNA AFTER purification -> colonies; Amp-resistance

plate 4: PCR-product (from K322127) + supD-tRNA BEFORE purification -> colonies; Amp-resistance

plate 5: K238013 + B0015 AFTER purification -> colonies; Amp-resistance

plate 6: K238013 + B0015 BEFORE purification -> colonies (half normal, half slightly red); Amp-resistance

plate 7: K228000 + RBS AFTER purification -> colonies; Amp-resistance

plate 8: K228000 + RBS BEFORE purification -> colonies; Amp-resistance

plate 9: PCR-product (from K322127) + supD-tRNA BEFORE purification [ligated with T4 ligase] -> colonies; Amp-resistance

picking occured with 200 µl pipette tip, thrown into 15 ml Falcon with 5 ml LB + antibiotic (1:1000)

shaking started at 18:00 p.m., 200 rpm, 37°C, o/n

MiniPrep

- miniprep (4 ml) of the 24 over-night cultures from 13-7-11:

2a-c: lacZ + T7-promotor BEFORE purification

3a-c: PCR-product (from K322127) + supD-tRNA AFTER purification

4a-c: PCR-product (from K322127) + supD-tRNA BEFORE purification

5a-c: K238013 + B0015 AFTER purification

6a-c: K238013 + B0015 BEFORE purification

7a-c: K228000 + RBS AFTER purification

8a-c: K228000 + RBS BEFORE purification

9a-c: PCR-product (from K322127) + supD-tRNA BEFORE purification [ligated with T4 ligase]

- measurement of the DNA concentration with nanodrop

2a: 25 ng/µl

2b: 215 ng/µl

2c: 9.5 ng/µl

3a: 85 ng/µl

3b: 53.5 ng/µl

3c: -

4a: 226 ng/µl

4b: 20 ng/µl

4c: 57.5 ng/µl

5a: 68.5 ng/µl

5b: -

5c: 20.5 ng/µl

6a: 360 ng/µl

6b: 143 ng/µl

6c: 305 ng/µl

7a: 443 ng/µl

7b: 218 ng/µl

7c: 204 ng/µl

8a: 228 ng/µl

8b: 268 ng/µl

8c: 218 ng/µl

9a: 38.5 ng/µl

9b: 16 ng/µl

9c: 16.5 ng/µl

Restriction

restriction digest of 19 plasmids from the miniprep (without 2c, 3c, 5b, 9b, 9c)

Restriction digest (total volume: 30 µl; DNA: 0,5µg; Enzyme (EcoR1 + Psi): 1 µl each; 3 µl NEB 4 buffer (10x); incubation: 50´@ 37°C)

Analytical agarosegel (1%, 1xTBE, 20 wells, 120 V, 400 mA, 50') for control of transformation success from 13-07-11:

10 µl sample + 2 µl loading dye (6x) -> 12 µl, 11 µl loaded on gel

5 µl 2-log DNA ladder

lanes:

0.) 2 log

1.) 2a -> lacZ + T7-promotor BEFORE purification

2.) 2b -> lacZ + T7-promotor BEFORE purification

3.) 3a -> PCR-product (from K322127) + supD-tRNA AFTER purification

4.) 3b -> PCR-product (from K322127) + supD-tRNA AFTER purification

5.) 4a -> PCR-product (from K322127) + supD-tRNA BEFORE purification

6.) 4b -> PCR-product (from K322127) + supD-tRNA BEFORE purification

7.) 4c -> PCR-product (from K322127) + supD-tRNA BEFORE purification

8.) 5a -> K238013 + B0015 AFTER purification

9.) 5c -> K238013 + B0015 AFTER purification

10.) 6a -> K238013 + B0015 BEFORE purification

11.) 6b -> K238013 + B0015 BEFORE purification

12.) 6c -> K238013 + B0015 BEFORE purification

13.) 7b -> K228000 + RBS AFTER purification

14.) 7a -> K228000 + RBS AFTER purification

15.) 7c -> K228000 + RBS AFTER purification

16.) 8a -> K228000 + RBS BEFORE purification

17.) 8b -> K228000 + RBS BEFORE purification

19.) 8c -> K228000 + RBS BEFORE purification

19.) 9a -> PCR-product (from K322127) + supD-tRNA BEFORE purification [ligated with T4 ligase]

PCR

Colony PCR for the ligation of K322127 and supD-tRNA. Therefore 15 colonies of the plates 3,4 and 9 (from 13-7-11) were resuspended in 50 µl nuclease free water. The experimet failed

Analytical agarosegel (1%, 1xTBE, 20 wells, 120 V, 400 mA, 90') for control of lthe colony-PCR from 15-7-11: 10 µl ligation sample + 2 µl loading dye (6x) -> 12 µl, 10 µl loaded on gel 5 µl 2-log DNA ladder

plate 3:

lanes:

1. 2-log

2. positive control

3-17. colonies from plate 3

18. positive control

plate 4:

lanes:

1. 2-log

2. negative control

3-17. samples from the colonies of plate 4

plate 9:

lanes:

1. 2-log

2-17. samples from colonies of plate 9

Restriction

Restriction digest of K238013+B0015 with S, P and K228000 (T7-Polymerase) + RBS with X, P

Restriction digest (total volume: 30 µl; DNA: 1 µg; Enzyme 1 µl each; 3 µl NEB 4 buffer (10x); incubation: 1 h @ 37°C)

The used DNA-templates were 8a (14-7-11) and 6c (14-7-11)

preparative agarosegel (1%, 1xTAE, 8 wells, 120 V, 400 mA, 90'):

30 µl restriction digest + 6 µl loading dye (6x) -> 35 µl loaded on gel

lanes:

1.) 2 log- DNA ladder 7 µl

2.) K22800 + RBS

3.) K238013 + B0015

4.) K322127 from an earlier restriction digest

-> DNA extraction from gel via mi-Gel-extraction kit from metabiom

Ligation

ligation of K228000-RBS with K238013-B0015 and K322127 with subD-tRNA.

Ligation (total volume: 30 µl; 10 µl of each DNA sample; 3 µl T4-ligase buffer (10x); 1 µl Quick-Ligase; 6 µl nf H20; 30`@ 37°C)

Transformation

transformation (40 µl competent cells; 1 µl DNA; Electroporation at 1510 V; incubation in Soc-Medium for 1 h @ 37°C, 50 µl on Amp-agar plate)

analytical agarosegel (1%, 1xTBE, 8 wells, 120 V, 400 mA, 90')

10 µl DNA + 2 µl loading dye -> 10 µl on gel

Lanes:

1.) 7 µl 2-log DNA ladder

2.) K228000-RBS + K238013 - B0015 (upper band in preparative gel)

3.) K228000-RBS + K238013 - B0015 (lower band in preparative gel)

4.) K322127-tRNA

Restriction

control restriction digest with the Enzymes E, P of 2b (=ligation product lacZ+T7 promotor), 6c (=ligation product K238013+B0015) and 8a (ligation product K228000+RBS).

Restriction digest (total volume: 30 µl; DNA: 0.5 µg; Enzyme 1 µl each (E, P); 3 µl NEB 4 buffer (10x); incubation: 1 h @ 37°C)

analytical gelelektrophoresis (1%, 1x TBE, 12 wells, 120 V, 400 mA, 90´)

lanes:

1.) 2b ( =ligation product lacZ+T7-Promotor)

3.) 6c (=ligation product K238013 B B0015)

5.) 8A (=ligation product K228000 + RBS)

6.) 2-log DNA ladder

MiniPrep

miniprep of the 4 ml overnight cultures from 21-7-11 with metabion

10 a-c: ligation product K322127 + tRNA

11 a-c: ligation product K228000-RBS + K238013+B0015

- nanodrop measurement of the DNA concentration:

10 a: 62 ng/µl

10 b: 42 ng/µl

10 c: 38.5 ng/µl

11 a: 51 ng/µl

11 b: 47.5 ng/µl

11 c: 47.5 ng/µl

Restriction

control regristiction digest of 10 a-c (ligation product K322127 + tRNA) and 11 a-c (ligation product K228000-RBS + K238013+B0015) with E, P

Restriction digest (total volume: 30 µl; DNA: 0.3 µg; Enzyme 1 µl each (E, P); 3 µl NEB 4 buffer (10x); incubation: 1 h 10` @ 37°C, inactivation 20´@ 80°C)

analytical gelelectrophoresis (1%, TAE 1x, 12 wells, 120 V, 400 mA, 120`)

lanes:

1.) 2-log ( 1 µl 2-log, 1 µl loading buffer (6x), 10 µl water) -> 10 µl loaded on gel

2.) 10 a

3.) 10 b

4.) 10 c

5.) 11 a

6.) 11 b

7.) 11 c