Ligation

Three ligations in total were conducted. K238013 (S,P) was quick-ligated with lacZ (X,P), rbs (S,P) with T7Pol (X,P) and T7Prom (S,P) with lacZ (X,P).

Transformation

The ligations were transformed into DH5alpha. Additionally, K238013 (from23-08-2011) was transformed into DH5alpha. The ligation of K238013 with lacZ is plated onto Amp and Cm, rbs with T7pol is plated onto Amp, T7Prom with lacZ onto Kan and K238013 is plated onto Amp and Cm.

Digestion

R2 (red writing: MiniPrep iGEM R2 31.5.11) was digested with X,P and T7prom (black writing: 17.6.11 T7) with S,P. After heat-inactivation, GLP was added to the digested DNA. The samples were applied onto a 1 % Agarose gel. The samples, along with those from the PCR (see below), were applied as follows: 2lod DNA ladder (5 ul); R2 (20 ul); R2 (20 ul); T7prom (20 ul); T7 prom (20 ul); PCR cut; positive control; PCR approach 1; PCR approach 1; PCR approach 2; PCR approach 2; PCR approaches 1 and 2 pooled. The gel was run for 1:30 h at 110 V. The following bands were expected: 3.1 kbp for lacZ with rbs and 2 kbp for vector pSB1A2. 3.4 kbp for T7prom in pSB1AK8. The bands are excised from the gel and the DNA is recovered using freeze 'n squeeze.

The original file before cutting of the bands seems to be corrupt.

Ligation

In the next step, K238013 (from 23-08-2011; cut with S,P) was ligated with rbs&lacZ (=R2; X,P), rbs (from 22-08-2011; S,P) with T7pol (from 23-08-2011; X,P) and T7prom (S,P) with rbs&lacZ (X,P). Each time, ligation was performed as normal and as quick-ligation. After that, the transformed cells were plated onto LB (Amp) plates and incubated over night.

New PCR of K322128 using Phusion High-Fidelity PCR-Kit (NEB/Finnzymes)

Material

BBa_K322127 with primers BBa_K568000, derived from Biomers, 3,8 kbp cut vector pSB1A2 digestion with X,P from Alex on 25.8.2011 (Florian 6.7.11 labelled with "K322127 PCR product aus gel ausgeschnitten")

Procedure

using same protocol as Florian on 11.7 

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

only 2 samples were prepared using each:

35,5µl of ddH2O 10 µl5x buffer 1 µl dNTPS 1 µl of template DNA K322127 (1,5ng/µl ) 1 µl of Primer fwd (10µM) 1 µl of Primer rev (10µM)

only the 10kB positive control was done:

34µl of ddH2O 10 µl5x buffer 1 µl dNTPS 1 µl of template DNA (template for positive control) 2,5 µl of Primer (10kB-Mix)

after pcr the samples were applied on a 1% Agarose-gel

The bands are excised from the gel and the DNA is recovered using freeze 'n squeeze.

two times 10µl of the PCR product were digested with X,P

the digestion and the pcr product were stored at -20°C

Ethanol precipitation of digested PCR product

Material: PCR product cut with X,P from the 24.8.2011 protocol see Methods the sample was applied on an agarose gel because the nanodrop indicated no DNA

Result

The gel showed that no DNA was yielded (see picture Restriction & Ligation)

Repeated Digest and Ligation of PCR Product

Material: PCR product of BBa_K322127 with primers BBa_K568000, derived from Biomers, 3,8 kbp cut vector pSB1A2 digestion with X,P from Alex on 25.8.2011 (Florian 6.7.11 labelled with "K322127 PCR product aus gel ausgeschnitten") Procedure: The PCR product was digested with X,P. After heat-inactivation, GLP was added to the digested DNA. The samples were applied onto a 0,8 % Agarose gel. The gel was run for 1:30 h at 110 V. The bands are excised from the gel and the DNA is recovered using freeze 'n squeeze. Ligation of the cut fragment with the vector pSB1A2 (cut with X,P; see Restriction & Ligation above) using quick ligation protocol see Methods

Result

Miniprep & Glycero-Stock: The cultures inoculated yesterday on LB were frozen at -80 °C as glycero-stocks. The culture of K238013 (pSB1A2) in DH5alpha was prepped. The resulting DNA yielded a concentration of approximately 300 ng/ul.

Repetition of transformations:

The transformations from yesterday (ligation using ligation protocol) were repeated using DH5alpha cells (kindly provided by Andrea). Transformations were done for reporter plasmid (part 2b) colony 5 from DATUM, K238013 with lacZ, rbs with T7pol and T7prom with lacZ. After incubation for one hour, the cells were plated on LB Amp (K238013 with lacZ; rbs with T7pol) and on LB Kan (reporter plasmid; T7prom with lacZ) and incubated over night.

Ligation of new PCR product with pSB1A2

Material:

cut PCR (X,P) product from K322127 from PCR 25.8.2011 cut pSB1A2 (X,P) from Alex from 25.8.

procedure:

we purified PCR product using Zymo DNA Clean +Concentrator 25

ligation:

sample 1: 15µl PCR product 5µl vector sample 2: 10µl PCR product 10µl vector

Transformation of the ligation and R2 as transformation control:

the two samples from purifiying and the part R2( OmpR in pSB1A2) as a positive control were transformed via electroperforation in competent DH5alpha cells that were kindly supplied by Andrea Mückl.

inoculation the following were inoculated: T7 Promotor with lacZ 2x K322127 in DH5alpha

Retransformation of reporter plasmid clone 5

the reporter plasmid clone 5 (118ng/µl) was diluted to 50 pg/µl and to 20 ng/µl

1 µl of each dilution was transformed into DH5alpha

Transformation of RBS

0,42 µl of RBS (47,5 ng/µl) were transformed into DH5alpha

Electrocompetent cells

new electrocompetent cells (DH5alpha) were made by the following protocol:

a 20 ml Luria medium overnight culture was inoculated with electrocompetent DH5alpha (kindly provided Andrea Mückl) yesterday

350 ml Luria medium were inoculated with 15 ml of the overnight culture and incubated at 37°C until a OD(600) of 0,7 was reached

the medium was distributed on falcons and the cells were centrifuged at 4500 rpm for 20 min at 4°C

after discarding the supernatant 350 ml of sterile ice-cold 10% glycerol was added and the pellets were resuspended using a pipet

the resuspension was centrifuged at 4500 rpm at 4°C for 11 minutes

after discarding the supernatant the cells were resuspended again in 350 ml of ice-cold 10% glycerol ( cells kept chilled all the time)

and again centrifuged at 4500 rpm at 4°C for 11 minutes

the supernatant was discarded and the cells were resuspended in the remaining supernatant

the cells were dispensed in 40 µl aliquots and stored at -80°C for further use

Planning

In order to get ligations working, purification steps will be performed using Promegas Wizard PCR and Gel clean up Kit. We are going to ligate PCR product into pSB1C3 and upstream of SupD-tRNA. In parallel, T7ptag will be ligated into AND-Gate Plasmid, which will be pSB1C3. [First step of assembly]

Then, synthesis product, which contains parts from SupDtRNA to RBS J44001, will be ligated into succesful AND-Gate Plasmid-T7ptag. The last step will be ligation of PCR-product into this construct.

Excel sheets can be found in DropBox folder "protocols".

planned parts

PCR product ligated into pSB1C3 will be a new part "red light sensor without lacZ".

PCR product ligated with SupD will be a new part as well.

Synthesis product is another maybe not so useful part which will be send into the registry.

Reporter plasmid will be another Part. This one was called "part 2B" in recent cloning steps and needs further validation (see 29.8. retransformation and sequencing)

Restriction digest

All digestions were complete and running on the expected lengths.

Purification of restricion digests from gel with Wizard Kit

the following items were purified using the Wizard Kit according to manufacturer's instructions and the concentration was determined by nanodrop:

1: 24,5 ng/µl

2: 24,5ng/µl

3: 24,5 ng/µl

5: 20,5 ng/µl

9: 20,0 ng/µl

10: 23 ng/µl

11: 21,5ng/µl

12: 29,5 ng/µl

13: 20,0 ng/µl

for more details see restriction digest table

Inoculation

the following overnight cultures were inoculated:

RBS reporter plasmid clone 1-5 K322127 from plate from 24.8.2011

Ligation

Minipreparation of DNA

The following concentrations were gained through minipreparation:

RBS  : 86,5 ng/µl

reporter plasmid clone 1: 135 ng/µl

reporter plasmid clone 2: 234 ng/µl

reporter plasmid clone 3: 102 ng/µl

reporter plasmid clone 4: 119 ng/µl

reporter plasmid clone 5: 96 ng/µl

The reporter plasmid clone 1-5 were sent to be sequenced at GATC.

gel purification of 30.8.11 digest

20 µl of remaining digestions (made on 30.8.11) were loaded on 1 % agarose gel with 1xTAE. Items 1,2,3,4,5,6,7,8,9,10,12 were used. This samples were cut using 350nm UV illumination as short as possible. Cut gel slices were subsequently purified using Promegas Wizard Kit according to manufacturers instructions.

Determination of concentration with Nanodrop resultet in not reliable data. Therefore quantitative 0.7 % agarose gel was run with 5 µl of each sample. 5 µl 2 log ladder was used (100 µg/ml). PCR Band (3.8 kbp) in line 2 was estimated to 75 ng. This gives aproximately 15 ng/µl. Intensities of 2 kbp band in line 4 as well as lines 5, 6, 7, 10 and 11 correspond to approx. 150 ng amount (--> 30 ng/µl).

Due to fly-by-night intensity of ladder those concentrations will be determined again on 02.09.11 for further ligations. Ligations today will be done with the following concentrations

o/n ligation with T4 Ligase

P, S opened Vector containing blue light promotor was ligated with RBS-LacZ insert. Due to huge insert lengths, insert:vector ratios were choosen as can be seen in ligation table.

Ligation was conducted at 16° C in thermocycler o/n using 20 µl sample volume and 1 µl T4 DNA ligase. Examplary calculation of I/V ratio for 6x molar excess of insert (source: http://openwetware.org/wiki/DNA_Ligation):

Digest of T7 promoter

Full volume of digest was loaded onto gel (50 µl digest + 10 µl 6 x Gel loading buffer)

Band with correct size of 3,5 kbp was cut using 365 nm illumination. Illumination time was kept as short as possible, picture was taken after cutting gel slice.

Gel purification of T7 promotor

Band was purified using Promega's Wizard Gel purification Kit according to manufacturer's instructions. Eluted DNA was subsequently quantified with an analytical 0.7 % agarose gel (120 V for 45 min)

Band in line 2 corresponds to 120 ng band of marker. Therefore concentration of purified T7 promoter is approx. 24 ng/µl. RBS_lacZ band is approx. 20 ng/µl and pSB1C3 band approx. 16 ng/µl

Ligation of Reporter Plasmid

digested T7 promoter was ligated with RBS_lacZ from 1.9.11 purification.

Ligation was conducted o/n at 16°C.

Precipitation of Ligations and Transformation

Todays Transformation were done after purifing ligation mix with Glycogen/Ethanol precipitation in order to increase transformed amount and purity of ligated vectors.

All Ligations of 02-09-11 and following items of 01-09-11 ligation were used:

1 (pos control pSB1A3single cut (10))

2 (PCR(1)-pSB1C3 linearized (3))

4 (T7ptag(1)-pSB1C3 (12))

6 (blue light(8) - lacZ(9))

Glycogen/ethanol precipitation:

-20 µl of the ligation mixed with 2 µl NaAc (3M), 0.5 µl glycogen and 22.5 µl isopropanol

-incubation step for 1 h @ -20 °C

-15 mins @ 10.000 rpms

-withdraw supernatant

-wash pelett with 70 % EtOH ~ 10 µl

-dry pelett and resuspense in 10 µl water

Transformation via electroporation was done with the whole cleaned 10 µl in 40 µl DH5alpha cells.

Ligations of PCR product into pSB1C3 and pSB1A3

Following ligations were examined using an excess of vector and 1 µl PCR-product (corresponds to approx 14 ng/µl).

Items 1+2 were incubated at room temperature and items 3+4 were incubated at 37° C for 1h each. 5 µl of ligations were subsequently electroporated into 40 µl undiluted DH5alpha cells.

Reporter Plasmid

As recent transformation of reporter plasmid yielded high vector background new preps will be done according to following scheme.

digest

long 1% preperative agarose gel and 3% preperative agarose gel have been produced and stored in a plastic bag with some 1x TAE buffer at 4°C o/n. Purification will be done tomorrow.

Minipreps of picked clones of Bluelight_lacZ 01-09-11 ligation (items 6+7)

[Flo]

Miniprep of 5 o/n cultures [names below protocol] (Metabion Kit)

All centrifugation steps were performed at 13.000 rpm and RT

1. Pellet 2 x 1,5 ml of bacterial suspension in one 2 ml-Eppi, centrifuge for 1 min each

2. Add 250 ul Bact.resuspension buffer, vortex

3. Add 250 ul Cell lysis buffer, invert 5x, incub. approx. 4 min

4. Add 350 ul DNA binding buffer, invert 5x, centrifuge for 10 min

5. Transfer clear lysate on column, centrifuge for 1 min, discard supernatant

6. Add 600 ul Column Wash Buffer, centrifuge for 1 min

7. Transfer column to new 1,5 ml-Eppi

8. Add 50 ul nuclease-free water (from Promega Wizard Kit), incub. approx. 2 min, centrifuge for 1 min

9. Store at -20°C

names:

-Blue Prom + lacZ #1 (= former "blue lacZ Trafo 7, Klon 2")

-Blue Prom + lacZ #2 (= former "blue lacZ Trafo 7, Klon 1")

-Blue Prom + lacZ #3 (= former "blue lacZ Trafo 6, Klon 1")

-Cph8 in DH5a

-K322127

Preparing glycerol stocks from cultures used above

Prepare 50% glycerol/LB solution by mixing 2 ml 100% glycerol and 2 ml LB

Add 300 ul of 50% glycerol/LB solution to 700 ul bacterial suspension to obtain an end concentration of 15% glycerol

Store at -80°C

Restriction digest of 5 Minipreps from today

Each plasmid has been cut with EcoRI HF AND EcoRI HF + PstI HF (= 2 digests per plasmid) as follows:

-5 ul Plasmid DNA

-2 ul 10x NEB buffer 4

-0.5 ul EcoRI HF

-(0.5 ul PstI HF)

-12 (11.5) ul ddH2O

end volume is 20 ul

mix, spin down, incub. at 37°C for 1 hour, inactivate at 80°C for 20 min

Add 4 ul 6x loading dye

Store at -20°C until tomorrow

--> quantification on gel 05-09-11

Analytical gel of Bluelight_lacZ, cph8 from Uppsala and K322127 testdigest

Size of bands look fine. Expected: 3093bp(lacZ)+86bp(Bluelight)= 3179 bp

--> Send clone 2 for sequencing or load RBS_lacZ digest along with ligated construct on gel and check for 86 bp difference.

cph8(2,2kbp) and K322127(4kbp) are running on their correct sizes.

Miniprep and testdigest of picked colonies on 04-09-2011

Miniprep of yesterdays picked clones were done. Concentration was determined using Nanodrop.

T7 Pol in pSB1C3 (ligation 4 from 1.9.)samples:

clone 1 : 58,5 ng/µl (sample 1)

clone 2 : 60,5 ng/µl (sample 2)

clone 3 : 55,5 ng/µl (sample 10)

clone 4 : 56,5 ng/µl (sample 13)

PCR in linearized pSB1C3:

clone 1 : 44,0 ng/µl (sample 4)

clone 2 : 82,0 ng/µl (sample 7)

clone 3 : 77,5 ng/µl (sample 5)

clone 4 : 70,0 ng/µl (sample 14)

blue light promotor+ lacZ (ligation 6 from 1.9):

clone 1 : 79,0 ng/µl (sample 12)

clone 2 : 67,0 ng/µl (sample 9)

clone 3 : 55,5 ng/µl (sample 21)

clone 4 : 65,0 ng/µl (sample 19)

red light sensor in pSB1C3 ratio 1:3

clone 1 : 58,0 ng/µl (sample 11)

clone 2 : 52,5 ng/µl (sample 15)

clone 3 : 59,5 ng/µl (sample 8)

red light sensor in pSB1C3 ratio 1:6

clone 1 : 50,5 ng/µl (sample 18)

clone 2 : 58,5 ng/µl (sample 3)

clone 3 : 61,5 ng/µl (sample 6)

red light sensor in pSB1A3 (ligation 1:5 (from 1.9.) clone 1 : 52,5 ng/µl (sample 20)(red colony) clone 2 : 51,0 ng/µl (sample 16)

red light sensor in pSB1A3 (ligation 1:1 (from 3.9) (red colony): clone 1 : 40,5 ng/µl (sample 17)

All samples were digested except 9,21,19:

5 µl of Mini-DNA was cut using 0,5 µl E and 0,5 µl P in 2 µl NEB4 and 20 µl total volume. Digest was loaded onto 1 % agarose gel.

Red light sensor constructs look fine (3,8kbp insert and 2 kbp vector). T7ptag constructs are wrong and will be repeated (T7ptag expected at 2,6 kbp). Blue light sensor looks fine (3kbp). Red light sensor cloning into pSB1A3 didnt yield any insertion products, mainly slightly red colonies were visible --> 1kbp insert is RFP cassette. One not-red clone could be picked (sample 15), which contains a 400 bp insert of unkown origin.

--> Sample 7 was choosen to be sequenced as it yielded most amount of DNA.

Reporter Plasmid: T7 promotor prep and new RBS_LacZ prep

3 % agarose gel from yesterday was used at 100V for 1,5h

No band could be detected. Nevertheless, gel slice was cut and will be purified.

Bands were cut and purified using Wizard Kit according to manufacturer's instructions.

Concentrations was determined using Nanodrop:

T7prom E, S: 9 ng/µl

T7prom P, S: 11.5 ng/µl

RBS_lacZ E, X: 14 ng/µl

RBS_lacZ E, X: 14.5 ng/µl

Reporter Plasmid

Purification of 05-09-11 gel runs

Frozen gel slices were purified using Promega's Wizard Kit according to manufacturers instructions.

Subsequent determination of concentration via Nanodrop:

1: T7prom 46 bp cut: 9 ng/µl

2: T7prom vector cut: 11.5 ng/µl

3: RBS_lacZ vector cut: 14.0 ng/µl

4: RBS_lacZ insert cut: 14.5 ng/µl

Ligation of purified samples: Reporter Plasmid and T7ptag into shipping vector pSB1C3

Ligation mix was incubated for 1h at RT and and then at 16°C o/n.

Reporter Plasmid and T7ptag into pSB1C3

Glycogen/ethanol precipitation and transformation

- DNA precipitation with glycogen:of the ligation samples from 6-9-11

1. add 2 µl 3 M sodium acetate, 0.5 µl glycogen and 22.5 µl isopropanol to 20 µl ligation product

2. incubate the mixture at -20°C for one hour

3. centrifuge the mixture for 15 minutes at 18,000 rpm, 4°C

4. discard the supernatant

5. add 50 µl 70 % ethanol

6. centrifuge the mixture for another 10 minutes at 18,000 rpm, 4°C

7. remove supernatant with a pipet CAREFULLY. If pellet moves, centrifuge again.

8. air-dry the pellet until the ethanol is completely evaporated. Do not over-dry pellet as this makes dissolving more difficult.

9. resuspend the pellet in 10 µl nuclease-free water by pipetting carefully.

• 10 µl purified ligation was mixed with 40 µl competent cells and electroporated into DH5alpha.
• 1 ml SOC medium (SOB medium + 20 mM glucose) was used and incubation was carried out for 1h at 37°C, shaking at 500 rpm in 2 ml eppis.
• Culture was then centrifuged at 4000 rpm for 5 minutes, and resolved in 100 µl medium.
• Whole sample was plated onto LB/agar plates supplemented with appropriate antibiotic and incubated o/n @ 37°C

Synthetic construct

Digest of synthetic construct and PCR-product (in pSB1C3)

Samples were loaded on preparative gel (1 %, TAE 1x, 100 V, 2 h 30 min) and subsequently cut with a new scalpel for each sample using 365 nm illumination as short as possible. Gel slices were purified using Promega's Wizard Kit according to manufacturer's instructions. Picture was taken after cutting of gel slices in order to reduce illumination.

ligation of synthetic construct and PCR-product (in pSB1C3)

Ligation was performed at 16°C o/n

AND-Gate plasmid

- DNA precipitation with glycogen/ethanol of the ligation samples from 7-9-11 accroding to protocol 7-9-11

- transformation via electroporation of 40 µl electro competent DH5alpha cells with the full volume of purified ligation samples from 7-9-11

Reporter plasmid and T7ptag

-overnight cultures for mini prep of transformations from 7-9-2011:

four clones of plates:

2 (T7prom(2)_RBS_lacZ(4))

3 (T7prom(2)_RBS_lacZ(4))

5 (RBS_lacZ(3)_T7prom(1))

6 (pSB1C3(3)_T7pol(5))

7 (pSB1C3(3)_T7pol(5))

one clone of plate 13 (synthetic construct in delivery vector pMA-RQ)

Reporter plasmid and T7 polymerase

- Plasmid isolation -> Miniprep of the over night cultures from 8-9-11 according to manufacturers instructions from Metabion

- measurement of the DNA concentration of the following samples:

2a: 92 ng/µl

2b: 122 ng/µl

2c: 131 ng/µl

3a: 98 ng/µl

3b: 152 ng/µl

3c: 124 ng/µl

3d: 112 ng/µl

5a: 52.5 ng/µl

5b: 55 ng/µl

5c: 74.5 ng/µl

5d: 87 ng/µl

6a: 134 ng/µl

6b: 118 ng/µl

6c: 101 ng/µl

6d: 140 ng/µl

7a: 76.5 ng/µl

7b: 113 ng/µl

7c: 83 ng/µl

7d: 71.5 ng/µl

13: 79 ng/µl

- control restriction digest of the 21 DNA samples (2a-d, 3a-d, 5a-d, 6a-d, 7a-d,13) with the restricition enzymes E,P

total volume 50 µl (39 µl nf H20, 5 µl DNA, 5 µl NEB4 Buffer, 0,5 µl of each enzyme)

incubation for 1 hour at 37°C, inactivation for 20 min at 80°C

- analytical gelelectrophoresis ( 1%, 1x TAE, 12 wells, 120 V, 1 hour 30 minutes)

50 µl DNA + 10 µl loading buffer -> 40 µl sample in each well

Size of T7promotor with RBS_lacZ looks correct (RBS_lacZ approx 3kbp pSB1AK8 approx 3.4 kbp). Ligation of 46 bp T7promotor into pSB1A2_RBS_lacZ (ID 5) didnt yield any correct clones, only vector background could be picked. Samples were discarded. Transformation of sample 4 resulted in short-circuit and was lost. Ligation of T7ptag into pSB1C3 looks fine. (T7ptag approx 2.6 kbp)

AND-Gate plasmid

transformation output: vector background looks fine

following clones have been picked:

AND-Gate plasmid

red_light sensor - synthetic construct ligation check

- Plasmid isolation -> Miniprep of the over night cultures from 09-09-11 according to manufacturers instructions from Metabion. 4 ml culture was used and 1 ml culture was saved for potential glycerol stocks

- control restriction digest of the 21 DNA samples (2a-d, 3a-d, 5a-d, 6a-d, 7a-d,13) with the restricition enzymes E,S

total volume 10 µl (7 µl nf H20, 1 µl DNA, 1 µl NEB4 Buffer, 1 µl of each enzyme)

nuke for 1 minute at 800 W (microwave)

- analytical gelelectrophoresis ( 0.7%, 1 x TAE, 12 wells, 160 V, 20 minutes)

10 µl DNA + 2 µl loading buffer -> 12 µl sample in each well

Sample ID 3c was choosen to be used for further cloning. pSB1C3 with red light sensor and synthetic construct

Nanodrop derived DNA conc. of 3c: 114 ng/µl

samples 2, 3a, 4b and 6a were discarded

sample 7b was choosen to be synthetic construct in pSB1C3 --> sequencing on monday

last step: assembly of redlight sensor_synthetic construct in pSB1C3 with T7ptag

we took 2 approaches: ligation of T7ptag as insert and vector, respectively

Digestions

samples were loaded on 1 % preparative agarose gel and run for 1.5 h at 120V

Samples were cut (365 nm illumination as short as possible, picture taken after cutting of gel slices) and purified using Promegas Wizard purification kit according to manufacturers instructions.

Ligations

Samples were incubated o/n at 16°C.

Glycogen/EtOH-precipitation of ligation samples from 10-09-11 and transformation

Put everything on ice before starting to work! Pre-cool centrifuge!

See protocol for precipitation/transformation from 07-09-11[See Methods]

Changes made:

• all solutions were icecold
• pellet was air-dryed for about 10 min
• autoclaved water was used instead of nuclease-free, due to quick subsequent use in the transformation
• resuspending of the bacterial pellet after transformation was difficult (tried pipetting and vortexing), because the wrong centrifuge has been used
• 1,5 ml-Eppis were used instead of 2 ml-Eppis for the shaking step at 37°C in SOC

Samples 1, 8 and 9: comp. cells used were DH5a from 01-07-11

Samples 2 - 7: comp. cells used were DH5a from 30-08-11

Antibiotic resistance:

• 1: Amp
• 2 - 9: Cam

Part I746907 (T7 promoter, GFP) in pSB1A2 was electroporated into DH5alpha, and plated onto a LB-Agar Amp plate. This part can be used as a reporter plasmid for our AND-Gate in e. coli CP919.

AND-Gate: Picking of colonies

Transformation and ligation efficiency of control was several thousand clones. Vector controls were lawn due to usage of CAM-plasmid contaminated electorcompetent DH5 alpha. All contaminated DH5alpha from 01-07-11 have been discarded.

3 clones of each plate have been picked and inoculated in 5 ml LB/cam o/n.

One colony from yesterday's transformation of Part I746907 in pSB1A2 was picked and inoculated into 5 ml LB Amp. Incubation o/n at 37 °C

AND-Gate

Minipreps of picked cones on 12-09-11 were done according to Metabion's instructions. Concentrations:

2a: c = 68.5 ng/µl

2b: c = 68.0 ng/µl

3a: c = 118 ng/µl

3b: c = 65.0 ng/µl

5a: c = 122 ng/µl

5b: c = 71.5 ng/µl

6a: c = 122 ng/µl

6b: c = 119 ng/µl

6c: c = 51.0 ng/µl

After restriction digest using E and P, samples were separated on a 0.8 % gel:

Vector prep pSB1A3 and pSB1C3

The restricted vectors ID10 pSB1A3 and ID12 pSB1C3 from 30-08-2011 were applied onto a 1 % agarose gel and prepped using the Wizard SV gel purification kit.

The vector pSB1A3 was single cut, while pSB1C3 was cut with E and P (from K322127). The expected lengths are about 2 kbp for pSB1C3 and about 3 kbp for pSB1A3, as it also contains a RFP cassette.

The measured concentrations were 12.5 ng/µl for pSB1A3 and 11.5 ng/µl for pSB1C3, in a resuspended volume of 50 µl.

The over night culture of part I746907 (pSB1A2) in DH5alpha was centrifuged and the supernatant was removed. Because of time reasons, the miniprep will be done tomorrow, the pellet was stored at -20°C.

Furthermore, parts BBa_K568002 (blue light sensor, RBS, LacZ) and BBa_K568003 (T7 promoter, RBS, LacZ) need to be transfered into shipping vector pSB1C3. Therefore, the parts are digested, the vector has already been cut with E and P.

Digests

Blue light sensor, RBS, LacZ in pSB1A2 (tube labelled: Miniprep 04-09-2011, clone 2, c=50 ng/µl) and T7 promoter, RBS, lacZ in pSB1AK8 (miniprep from 09-09-2011, clone 2c, c=131 ng/µl) were cut with E and P.

Next, the digested DNA was loaded onto a preparative 1% gel. After the electrophoresis was complete, the correct bands were cut out on the UV transilluminator (365 nm) before the following gel photo was recorded:

The gel looks fine. Cutting out the correct band had to be done very carefully in lanes 1 and 2, because the bands for the vector and the insert were very close together (vector: 3.4 kb, insert: 3.1 kb). Lanes 3 and 4 also show the expected bands, at about 3.2 kb for the insert and about 2 kb for the vector.

Finally, the DNA was extracted and purified from the cut-out gel slices using the Promega Wizard Kit according to the kit protocol.

The following concentrations were measured (Nanodrop):

blue light sensor, RBS, lacZ: 10 ng/µl

T7 promoter, RBS, lacZ: 10 ng/µl

Ligations

As a next step, the following ligation was performed (see table below). We used the opportunity to compare the old (NEB) and the new (Metabion) ligase. The ligation was incubated at 16 °C over night.

Miniprep

Part I746907 (pSB1A2) was purified from yesterday's DH5alpha-pellet using the miniprep-kit according to protocol. The concentration was determined to be 63 ng/µl using Nanodrop.

Glycogen/Ethanol precipitation and Transformation

Yesterday's ligations were purified using glycogen/ethanol precipitation. The yielded 10 µl DNA-Solution were used completely for Transformation in DH5alpha. After electroporation, the cells were incubated at 37 °C for 1h in SOC-media. After this, they were plated onto LB-Agar plates with the appropriate antibiotics.

Digestions

The cloning of our last unfinished part (BBa_K568004), "optogenetical AND-Gate without first promoter - choose your first input" was started today. The part consists of our synthetic construct plus t7ptag. Our synthetic construct (in pMA-RQ) was digested for preparation using E and S, while the miniprepped part I746907 was digested using E and S to check if the prepped plasmids are correct (a mistake was made during preparation of the digest, so a second digest was made at the same time. Both digests were loaded onto the analytical gel):

The product of the preparative digest was loaded onto a preparative 1% agarose gel. After electrophoresis, the desired band was cut out on the UV-transilluminator (365 nm) before the gel photo was recorded:

The product of the preparative digest was loaded onto a preparative 1 % agarose gel. After electrophoresis, the desired band was cut out on the UV-transilluminator (365 nm) before the gel photo was recorded: :

The gel looks as expected. The cut out bands around 400 bp are the synthetic construct while the vector can be seen at 2.4 kb.

The product of the control digest was loaded onto an analytic 0.8 % agarose gel. The following gel photo was recorded:

Lane 1 (correct digest): Part BBa_I746907 is 924 bp long, the gel shows the corresponding bands. The vector pSB1A2 is 2079 bp long and can also be seen at the expected spot.

Lane 2 (wrong digest): The bands show that the digest doesn't work properly in 10x NEB4 buffer. The band around 3 kb is uncut or singly cut vector with insert.

Picking of clones

3 colonies were picked each from plates 2, 3 (Part BBa_K568003 in pSB1C3) and 4, 5 (Part BBa_568002 in pSB1C3) and then inoculated in 5 ml LB Cam each. The cultures were incubated at 37°C over night for miniprep tomorrow.

Cloning of Part BBa_K568004

To ligate the synthetic construct (cut yesterday with E, S) with T7ptag in pSB1C3, the following digestion was done:

After the digestion was completed, the sample was loaded onto a preparative 1 % agarose gel. After electrophoresis at 120 V, the correct bands were cut out on the transilluminator (365 nm, make sure UV lamp isn't switched on longer than absolutely necessary). Subsequently, the following gel photo was recorded:

pSB1C3 with T7ptag is 4743 bp long, which corresponds with the location of the cut-out band.

As a next step, the DNA was purified from the gel slice using the Promega Wizard Kit. The concentration of the product was determined to be 6 ng/µl using Nanodrop. This value is not reliable, but for lack of a second, quick way to measure the DNA concentration, the following ligation was perpared using the value nevertheless. Incubation of the Ligation occurred over night at 16 °C.

The product of yesterday's ligation was purified using glycogen-ethanol-precipitation (see methods). The product was stored at -20°C for transformation tomorrow.

Minipreps of parts BBa_K568003 and BBa_K568002

Yesterday's overnight cultures were prepped today according to protocol. A total of 12 cultures was prepped, 6 of which contained pSB1C3 with K568003 while the other 6 contained pSB1C3 with K568002. After the miniprep, the following concentrations were measured using Nanodrop:

Part K568003 (T7prom, RBS, lacZ):

• 2a: 86.5 ng/µl
• 2b: 71.0 ng/µl
• 2c: 63.5 ng/µl
• 3a: 66.5 ng/µl
• 3b: 88.5 ng/µl
• 3c: 85.0 ng/µl

Part K568002 (blue light promoter, RBS, LacZ):

• 4a: 82.0 ng/µl
• 4b: 78.0 ng/µl
• 4c: 80.0 ng/µl
• 5a: 74.5 ng/µl
• 5b: 97.0 ng/µl
• 5c: 100 ng/µl

Restriction digest and control gel

To check whether the prepped DNA samples contained the correct parts, the following digestion was performed:

After the digest was completed, the samples were loaded onto a 0.8% agarose gel. After electrophoresis, the following gel photo was recorded:

All bands look fine. Samples 1-6 correspond to the minipreps of K568003. Here the insert is 3147 bp and the vector is 2070 bp long. The bands verify this. Samples 7 - 12 correspond to the minipreps of K568002, which has an insert of 3187 bp and the same 2070 bp vector. Again, the bands show that this is correct.

Glycogen/ethanol precipitation

Yesterday's ligation was purified using glycogen/ethanol precipitation. The yielded DNA samples were used for transformation

Transformations

The transformations listed below were carried out. After electroporation the cells were incubated in 1 ml SOC medium for 1 h at 37 °C before plating.

For Redlight Sensor Characterization

All plasmids were electroporated into the new electrocompetent CP919 cells for characterization of the red light sensor later this week.

• Plate Number 1: Part K322127 in pSB1C3 (tube label "K322127 miniprep 04.09.2011"). Plated onto LB Cam plate.
• Plate Number 2: Part K568000 (tube label "5.9.11, 2 vom 1.9, redlight in pSB1C3, 83 ng/µl"). This is our PCR product (redlight sensor). Plated onto LB Cam plate.
• Plate Number 3: cph8 in pSB1A3 (tube label "cph8 Miniprep 4.9.11"). Plated onto both LB Amp.

For generation of Part BBa_K568004

The purified products of yesterday's ligation were electroporated into DH5alpha and plated onto LB Cam plates.

• Plate Number 4: Ligation 1 (16-09-2011), pSB1C3 T7ptag with synthetic construct, ligation ratio I:V = 5.
• Plate Number 5: Ligation 2 (16-09-2011), pSB1C3 T7ptag with synthetic construct, ligation ratio I:V = 10.
• Plate Number 6: Ligation 3 (16-09-2011), pSB1C3 T7ptag with synthetic construct, ligation ratio I:V = 15.
• Plate Number 7: Ligation 4 (16-09-2011), vector background pSB1C3 T7ptag.

The purified products of the ligation performed on 15-09-2011 were electroporated into DH5alpha, just to make sure that the part is really contained in pSB1A3 and not pSB1C3. Therefore, the transformed cells were plate onto LB Amp and LB Cam plates. The plate Number refers to the LB Amp plate as well as the LB Cam plate in all cases.

• Plate Number 8: Ligation 1 (15-09-2011), positive control pSB1A3 single cut
• Plate Number 9: Ligation 2 (15-09-2011) pSB1A3 T7ptag (purify 1.9.) with synthetic construct, ligation ratio I:V = 5.
• Plate Number 10: Ligation 3 (15-09-2011) pSB1A3 T7ptag (purify 1.9.) with synthetic construct, ligation ratio I:V = 10.
• Plate Number 11: Ligation 4 (15-09-2011) pSB1A3 T7ptag (purify 1.9.) with synthetic construct, ligation ratio I:V = 15.
• Plate Number 12: Ligation 5 (15-09-2011) vector background pSB1A3 T7ptag (purify 1.9.)

For cloning of I746907 into pSB6A1

We want to use I746907 as a reporter part for our optogenetical AND-gate in e. coli CP919. Therefore the part must be transformed into a low copy vector, since the AND-gate is in pSB1C3 which is a high copy vector. Because CP919 already has a Kan-Resistance, the low copy vector used for this needs to carry Amp-Resistance. That's why we chose pSB6A1, a low copy vector with Amp-Resistance. The DNA was gained from 2011 Kit Plate 1 well 1K by resuspension in 10 µl ddH2O. This was then used for electroporation into DH5alpha which were plated onto LB Amp plates after transformation.

• Plate Number 13: pSB6A1, 0.5 µl DNA used for electroporation
• Plate Number 14: pSB6A1, 1.0 µl DNA used for electroporation
• Plate Number 15: pSB6A1, 8.5 µl DNA used for electroporation

For Testing of I746907

To be able to characterize part I746907, we used a chemical transformation into BL21 DE3 (see methods). 1.5 µl of the DNA were used (c = 63 ng/µl). The transformed cells were plated onto a LB Amp plate.

• Plate Number 16: part I746907 in pSB1A2

Picking of clones of Bba_K568004 in pSB1C3 for miniprep

3 clones each were picked from plates 4, 5 and 6 (transformation from 17-09-2011) and 5 ml LB + Cam were inoculated and vortexed shortly for equal distribution of bacterial cells.

Falcon tubes were set almost horizontally in the 37°C-incubator to maximize oxygen input and shaking occurred @ 250 rpm for 3 h and 180 rpm for another 4.5 h.

Miniprep of 3 x 3 clones of Bba_K568004 in pSB1C3

Miniprep done according to kit instruction (Metabion "mi-plasmid mini prep") [See Methods]

Changes:

• All 5 ml bacterial suspension were used for the prep
• Autoclaved water for elution was pre-warmed to ~60°C

Names (according to plate # from 17-09-2011):

• K568004_4-1, c = 64.0 ng/µl
• K568004_4-2, c = 92.0 ng/µl
• K568004_4-3, c = 62.0 ng/µl
• K568004_5-1, c = 67.0 ng/µl
• K568004_5-2, c = 76.5 ng/µl
• K568004_5-3, c = 74.5 ng/µl
• K568004_6-1, c = 75.5 ng/µl
• K568004_6-2, c = 61.5 ng/µl
• K568004_6-3, c = 74.5 ng/µl

Part BBa_K568004

The 9 prepped samples of BBa_K568004 have to be checked for correct sizes of vector and insert. The following digestion was prepared:

Due to time reasons, 10 µl of the sample was digested for 1 min in the microwave at 800 W, while the other 10 µl of the sample were incubated at 37 °C.

The product of quick digest in the microwave was separated using electrophoresis with an analytical 0.8 % agarose gel. This yielded the following picture:

The bands of all samples look fine. The insert BBa_K568004 is 3069 bp long, the pSB1C3 vector is 2070 bp long. All samples show these bands. We chose sample 8 for shipping, because it looked "cleanest" on the gel.

The product of the "normally" incubated restriction digest (1 h at 37 °C) was also loaded onto an analytical 0.8 % agarose gel. The subsequent electrophoresis resulted in the following picture:

Again, the bands are at the expected positions.

Miniprep of pSB6A1

All six overnight cultures of pSB6A1 in DH5alpha were miniprepped according to metabion's protocol. The cell pellets were pink, indicating that the cells contained the desired plasmid, which carries an RFP-coding sequence as a standard insert. Due to a mistake, the pellet of one sample was lost.

The concentrations were measured using Nanodrop:

• Sample 1: c = 93.0 ng/µl
• Sample 2: c = 79.0 ng/µl
• Sample 3: c = 77.5 ng/µl
• Sample 4: c = 86.5 ng/µl
• Sample 5: c = 102 ng/µl

To be able to test the optogenetical AND-Gate in CP919, we need to transform the desired reporter part into a low copy vector. Therefore, BBa_ I746907 will be ligated into pSB6A1.

Digest

The following digest was performed:

After this, the samples were separated by electrophoresis using a preparative 1 % agarose gel. The correct gel bands were cut out on the transilluminator at 365 nm (keep irradiation time as short as possible). After this, the following photo was recorded:

The bands match the expectations.

Samples - : The cut out band around 4 kb is the cut vector pSB6A1, which is 4022 bp long. The band visible at around 1 kb is the RFP coding sequence (1069 bp).

Samples - : Part I746907 is 924 bp long. This corresponds to the cut out bands. The vector pSB1A2 can be seen at 2 kb (it is 2079 bp long).

Ligation

After purification of the DNA from the gel slices using the Promega Wizard Kit, the following ligation was prepared and incubated at 16 °C over night: