In Project: Team Sensor With tags: fapR, Malonyl-CoA ...2018.igem.org/wiki/images/3/32/T--Marburg... · Project: Team Sensor Page 5 Step Temperature Time 1. initial denaturation 98°C

Page 1Project: Team Sensor

created: 17.05.2018 19:00updated: 17.05.2018 19:13

Author: Benjamin DanielEntry 1/42: fapR insertet into entry Vector by golden gate ligationIn Project: Team SensorWith tags: fapR, Malonyl-CoA, Sensor, entry vector

In order to insert the fapR gene into our level 1 entry vector we performed a golden gate ligation using BsmBI and T7 ligase.

The ratio of insert to vector was 5:1. The vector had a concentration of 70 ng/µL and the insert 28.2 ng/µL.

The Vector had a size of 2.6 kB, while the insert was 0.56 kB.

Therefore 2.7 µL of the insert had to be used for 1 µL of vector to archive the 5:1 ratio.

Total Volume 10 µL

Insert 1 µL

Vector 2.7 µL

T4 ligase buffer 10X

1 µL

BsmBI 0.5 µL

T7 ligase 0.5 µL

H 02 4.3 µL


created: 24.05.2018 16:07updated: 25.05.2018 13:59

Author: Benjamin DanielEntry 2/42: Extraction of genomic DNA from the Pseudomonas putida strain KT2440In Project: Team SensorWith tags: Pseudomonas putida, 3HP, Sensor


A single colony of Pseudomonas putida KT2440 was picked, from which a culture was grown overnight at 30°C in LB media.

For the extraction of genomic DNA from , the DNeasy Blood & Tissue Kit from Qiagen was used. The extraction was P. putida

carried out twice in parallel.

1. Harvest cells (maximum 2 x 109 cells) in a microcentrifuge tube by centrifuging for 10 min at 5000 x g (7500 rpm). Discard supernatant.

2. Resuspend pellet in 180 µl Buffer ATL

3. Add 20 µl proteinase K. Mix thoroughly by vortexing, and incubate at 56°C until the tissue is completely lysed. Vortex occasionally during incubation to disperse the sample. Lysis is usually complete in 1–3 h. If it is more convenient, samples can be lysed overnight.

4. Vortex for 15 s. Add 200 µl Buffer AL to the sample, and mix thoroughly by vortexing. Then add 200 µl ethanol (96–100%), and mix again thoroughly by vortexing. It is essential that the sample, Buffer AL, and ethanol are mixed immediately and thoroughly by vortexing or pipetting to yield a homogeneous solution. Buffer AL and ethanol can be premixed and added together in one step to save time when processing multiple samples.

5. Pipet the mixture (including any precipitate) into the DNeasy Mini spin column placed in a 2 ml collection tube (provided). Centrifuge at 6000 x g (8000 rpm) for 1 min. Discard flow-through and collection tube.

6. Place the DNeasy Mini spin column in a new 2 ml collection tube (provided), add 500 µl Buffer AW1, and centrifuge for 1 min at 6000 x g (8000 rpm). Discard flow-through and collection tube.

7. Place the DNeasy Mini spin column in a new 2 ml collection tube (provided), add 500 µl Buffer AW2, and centrifuge for 3 min at 20,000 x g (14,000 rpm) to dry the DNeasy membrane. Discard flow-through and collection tube. It is important to dry the membrane of the DNeasy Mini spin column, since residual ethanol may interfere with subsequent reactions.

8. Place the DNeasy Mini spin column in a clean 1.5 ml or 2 ml microcentrifuge tube and pipet 200 µl Buffer AE directly onto the DNeasy membrane. Incubate at room temperature for 1 min, and then centrifuge for 1 min at 6000 x g (8000 rpm) to elute

The yields were 62ng/µL and 38.5ng/µL respectively, as measured via nanodrop.


created: 25.05.2018 13:59updated: 25.05.2018 16:29

Author: Benjamin DanielEntry 3/42: Amplification of the 3-HP sensor casette from Pseudomonas putida KT2440 chomosomal DNAIn Project: Team SensorWith tags: 3-HP, Sensor, PCR, Genomic DNA, Pseudomonas putida

To amplify the 3-HP sensor cassette from the chromosomal P. putida DNA, the primers oiGEM2501_f and oiGEM2502_r were used. The template had a concentration of 62 ng/µL.

Total: 50 µL

Buffer 5X 10 µL

Primer forward oiGEM 2501:

1 µL

Primer reverse oiGEM 2502:

1 µL

Template: 2.5 and 10 µL

dNTPs 1 µL

Polymerase 0.5 µL

DMSO 1.6 µL

H 02 33 and 25 µL

The protocol of the thermocycler


Step Temperature Time

1. initial denaturation

98°C 30 s

2. denaturation 98°C 10 s

3. annealing 61°C 40 s

4. extension 72°C 90 s GoTo 2. repeat 30 times

5. final extension 72°C 300 s

6. hold 4°C 30 s

The µL of the resulting product were run on a 2% agarose gel. Cyber green was used to stain the DNA.

The lanes 4 and 5 contain the products of the amplification for the 3-HP sensor. The asterix denote the expected running P. putida

hight. It cn not be seen on this gel, weather the band is of the expected size, due to the poor resolution in the target size. The run will be repeated with a lower concentration of agarose.

Unbenannt.JPG


created: 25.05.2018 14:16updated: 25.05.2018 16:26

Author: Benjamin DanielEntry 4/42: Annealing and finalizing of the FapO operatorIn Project: Team SensorWith tags: Fap, PCR, Sensor, Malonyl-CoA

To create the FapO operator, the two oligos oiGEM2504 and oiGEM2505 were combined using the following PCR protocoll:

Total: 50 µL

Buffer 5X 10 µL


0.5 µL


0.5 µL

dNTPs 1 µL

Polymerase 0.5 µL

DMSO 1.6 µL

H 02 38 µL



98°C 30 s

2. denaturation 98°C 10 s



5. final extension 72°C 300 s

6. hold 4°C 30 s

The µL of the resulting product were run on a 2% agarose gel. Cyber green was used to stain the DNA.

The gel picture can be seen below. Lane 1 contains the neb 100 bp purple ladder. Lane 2 and 3 contain the two PCR products for the fapO operator.

No bands are visible in the expected range of 60-80 bp. The PCR for the annealing will be repeated.


iGEMFapO+Putida_-_IM000267.tif


created: 25.05.2018 14:22updated: 25.05.2018 15:31

Author: Benjamin DanielEntry 5/42: Transformation of E.coli with the entry vector containing FapRIn Project: Team SensorWith tags: fapR, Fap, E. coli, entry vector, 3-HP, e.coli

Follows the entry: fapR insertet into entry Vector by golden gate ligation - entry #1 in project 'Team Sensor' (Benjamin Daniel, 17.05.2018)

The whole 10 µL of the product from the golden gate ligation of the FapR into the entry vector were used to transform E. coli neb turbo.

The as control, untransformed E. coli was used. On plate 1 and 5 E. coli was transformed FapR but no selection was used on plate 1, where a lawn of cells has grown. On plate 5 36µg/mL chloramphenicol was used for selection and only single colonies were observed. Plate 2 and 6 were transformed with PYTK as a positive control. They grew to a lawn without selection and to single colonies that clearly visible expressed GFP. Plate 3 and 4 did undergo transformwation procedure without any plasimid and were plated on selection and non-selection plates. Without antibiotic a lawn grew, without nothing. Since all controls show the expected result, everything is expected to have gone right.

Two single colonies from the fapR plates were picked and inoculated into 3 mL LB, incubated overnight and the plasmid extracted. This was then sent for sequencing.

20180522_081817.jpg

https://eln.labfolder.com/eln/notebook#?projectIds=76753&blockId=2492968

https://eln.labfolder.com/eln/notebook#?projectIds=76753&blockId=2492968


created: 28.05.2018 14:50updated: 28.05.2018 15:58

Author: Benjamin DanielEntry 6/42: Annealing and extension of fapO from B. subtilis 168In Project: Team SensorWith tags: Sensor, Malonyl-CoA, B.subtilis, fapO, Oligo

The second approach to the completion of the fapO operator for the Malonly-CoA sensor.

This time the PCR 2x Phanta Max Master Mix from Vazyme was used. It contains dNTPs, the Phanta Max Super-high-fidelity polymerase and buffers.

The reaction mix was as follows:

Total: 50 µL

PCR 2x Phanta Max Master Mix

25 µL


2 µL


2 µL

H 02 21 µL



95°C 30 s

2. denaturation

95°C 15 s



5. final extension

72°C 90 s

6. hold 4°C endless


10 µL of the PCR products of then run on a 3% agarose gel. The picture after 3 h is below. The one band indicated by the asterix is presumably the wanted fapO operator.

fap_o_2._Approach.tif


created: 28.05.2018 15:08updated: 05.06.2018 18:00

Author: Benjamin DanielEntry 7/42: mplification of the 3-HP sensor from Pseudomonas putida KT2440In Project: Team SensorWith tags: 3-HP, Sensor, Genomic DNA, PCR, Pseudomonas putida

The second approach to the amplification of the 3-HP sensor from Pseudomonas putida KT2440 by PCR on chromosomal DNA.

This time the PCR 2x Phanta Max Master Mix from Vazyme was used. It contains dNTPs, the Phanta Max Super-high-fidelity polymerase and buffers.

The reaction mix was as follows:

Total: 50 µL


25 µL

Template Genomic DNA 60ng/yL

3 yL

Primer forward oiGEM 2501_f:

2 µL

Primer reverse oiGEM 2502_r:

2 µL

H 02 18 µL



95°C 30 s

2. denaturation

95°C 15 s



5. final extension

72°C 180 s



The product from that PCR was run on a 1% agarose gel. The picture is shown below.

A strong band can be seen between 1 and 1.5 kb which corresponds well to the anticipated position since the product should be 1,159 bp long.

The Product was then purified by gel electrophoresis and subsequent excision. Thereafter, it was inserted into the entry vector 2 by golden gate ligation.

This was

P._putida_2.tif


created: 06.06.2018 16:13updated: 06.06.2018 16:24

Author: Benjamin DanielEntry 8/42: Second attempt on creating FapO from primers by annealing and extendingIn Project: Team SensorWith tags: Fap, 3-HP, Malonyl-CoA, Oligo, PCR, Sensor, B.subtilis

Total: 50 µL


25 µL

Primer forward oiGEM 2507_f:

2 µL

Primer reverse oiGEM 2508_r:

2 µL

H 02 18 µL



95°C 30 s

2. denaturation

95°C 15 s


5. final extension

72°C 180 s


The Primers were amplified using the protocol above. The result of the PCR was loaded onto a 3 % agarose gel stained with cyber green.

The resulting picture is below. The bright band at approximately 100bp is the sought after product. It was excised and the DNA was purified using the Thermo Fischer Gene Jet DNA extraction Kit.

A concentration of 9.3 ng/µL was detected by nano-drop. The DNA will be inserted into a level 0 entry vector by golden gate ligation.


FapO_2.Tif


created: 28.06.2018 15:21updated: 28.06.2018 15:39

Author: Benjamin DanielEntry 9/42: Level 0 FapOIn Project: Team SensorWith tags: fapO, E. coli, Malonyl-CoA, Sensor, B.subtilis

This describes the golden gate ligation of the FapO operator site into the entry vector oiGEM1002 to create piGEM2503.

The ingredients were combined and run in a thermocycler at the parameters given below. The resulting product was used to transform chemo-competent NEB turbo. After overnight incubation on Chloramphenicol plates, a dense mass of colonies could be seen. Of these only two were expressing GFP. Two of the correct colonies were selected and grown in CM liquid LB media. From this the Plasmid will be extracted and sent for sequencing.

Amount Compound

0.5 µl BsmB1

0.5 µl t7 Ligase

0.5 µl T4 Ligase Buffer

2.5 µl Entry Vector oiGEM1002 62.1 ng/µL

1 µl FapO amplificate 32.4 ng/µL

Time Temperature

2 min 42°C

5 min 16°C Goto: 1, repeat 30 times

30 min 60°C

Hold 4°C


created: 05.07.2018 16:29updated: 05.07.2018 16:50

Author: Benjamin DanielEntry 10/42: Assembly of the Level 1 plasmid piGEM2505 carrying the 3-HP Sensor and piGEM2508 carrying the malonyl CoA sensorIn Project: Team SensorWith tags: 3HP, 3HP, 3HP, 3HP, 3HP, Sensor, Sensor, Sensor, Sensor, 3HP Sensor, Pseudomonas putida, Pseudomonas putida, Pseudomonas putida, Pseudomonas putida, Pseudomonas putida

In order to create the level 1 plasmids, all necessary level 0 parts were gathered. The parts were added in equal molarity, except for the resistance part, which was added in 3x excess. (see table Calculations for level 1 assembly).

The mixture was run in a thermocycler at the following settings ( Table Thermocycler Settings)

Unbenannt.JPG


Calculations for Level 1 assembly

A B C D

Sheet1

calculations_for_level_1_assembly_2650895_3.xlsx

3 HP Sensor GFP piGEM 1011 Sensor Part piGEM2501 piGEM1042 piGEM10355´Connection Dummy 3 HP sensor sfGFP B0015

ng/µL 85.7 168 238 167bp 2131 3234 2820 2240(copy number*10^9)/µL 37.3 48.1 78.2 69.1Volume in µL 0.5 0.4 0.3 0.3

Malonyl-CoA Sensor GFP piGEM 1011 Sensor Part piGEM10425´Connection Dummy FapR FapO sfGFP

ng/µL 85.7 184 164 238bp 2131 2662 2172 2820(copy number*10^9)/µL 37.3 64 70 78.2Volume in µL 0.5 0.3 0.3 0.3

123456789

10111213141516171819202122

https://eln.labfolder.com/eln/workspace/block/2650895/3/downloadTableVersion


Reaction Mix

A B C D E F G

Sheet1

reaction_mix_2650895_4.xlsx

Reaktionmix in µL 2505 Reactionmix in µL 5.24 2508H20 5.42 H20 5.24T4 Ligase Buff (10X) 1.5 T4 Ligase Buff (10X) 1.5T7 Ligase 0.25 T7 Ligase 0.25BsaI 0.25 BsaI 0.25DNA 7.58 DNA 7.76Total 15 Total 15

123456789

1011121314151617181920



Thermocycler Settings

A B C D E F G H

Sheet1

thermocycler_settings_2650895_5.xlsx

Step Temperature Time1 37°C 2 min

2 16°C 5 minGoto 1: repeat 35x

3 37°C 30 min4 80°C 10 min

123456789

1011121314151617181920



created: 10.07.2018 16:30updated: 10.07.2018 16:44

Author: Benjamin DanielEntry 11/42: Protokoll for the assembly of the Level 1 sensor partsIn Project: Team SensorWith tags: 3-HP, 3HP Sensor, B.subtilis, Fap, fapO, Sensor, Pseudomonas putida, 3HP, fapR

2 minIn order to calculate the necessary amounts of the different parts to make them equimolar a excelsheet was built. The copy number of the different level 0 parts was calculated using the following formula:

copy number = (ng*6.0022*10^23)/((length in bp)*650*10^9)

All parts were combined in the calculated amounts, with 1/3 of the resistance part, and the thermocycler was run at the following protocol:

Time Temperature

2 min 37°C

2 min 16°C

Goto step 1, repeat 60X

60 min 80°C

10 min 60°C

Hold 4°C

The result was transformed into chemocompetent E. coli NEB turbo cells. These were regenerated in LB media for 1h and plated on LB Kan 25µg/mL plates

After overnight incubation, no colonies were observed.

This is probably due to a lack in the quality of the purified resistance part. The experiment will be repeated, once the part is freshly purified.


Unbenannt.JPG


created: 10.07.2018 16:48updated: 10.07.2018 16:55

Author: Benjamin DanielEntry 12/42: Predigestion of piGEM1057 and piGEM1056 by BsaI.In Project: Team SensorWith tags: level 0, Sensor

Predigestion of piGEM1057 and piGEM1056 by BsaI. An overnight culture of E. coli transformed with piGEM1057 and piGEM1056 was harvested via miniprep. The result was 35µL of 247ng/µL forp iGEM1057 and 213ng/µL for piGEM1056.

With this, an digestion was started.

Ingriedient Amount in µL

Plasmid 21.5

CutSmart 10x Buffer

2.5

BsaI 1 µL

The Plasmid was digested overnight at 37°C. Then it was purified using gel electrophoresis in a 1% agarose gel. The correct bands were cut from the gel and purified using the GeneJet Gel purification kit from ThermoFischer.


created: 13.07.2018 08:55updated: 13.07.2018 09:10

Author: Benjamin DanielEntry 13/42: Transformation of chemocompetent E. coli with Level 1 plasmids for Malonyl-CoA and 3-HP sensorsIn Project: Team SensorWith tags: transformation, chemokompetent, E. coli, 3HP Sensor, Sensor, Malonyl-CoA, level 1

For the transformation:

1. Take the aliquot of chemocompetent E. coli cells from the -80°C freezer and thaw on ice for about 10 minutes.

2. Add the plasmid and mix by stirring with the pipette tip (no pipetting up and down)

3. Keep on ice for another 20-30 minutes

4. Heatshock at 42°C for 45 seconds

5. Quickly place on ice to cool down for 2-3 minutes

6. Add 500µL regeneration media (LB1, Sock, YT, TB) (NO ANTIBIOTIC)

7. Incubate at 37°C for 1-2 hours (depending on the antibiotic used, 1 hour for Chloramphenicol, 1.5 hours for Kanamycin)

--> Take the plates you want to plate on oout of the fridge, so they are at room temperature when you plate.

8. Gently centrifuge at 6,000 rpm for 1 minute

9. remove 430 µL of the supernatant, then resuspend the pellet in the remaining media (by pipetting up and down)

10. plate on agar plates with the appropriate antibiotic

11. incubate at 37°C overnight


created: 16.07.2018 13:25updated: 25.07.2018 13:56

Author: Franziska NouschEntry 14/42: miniprep and digest of 3HP-GFP SensorIn Project: Team SensorWith tags: Sensor, 3HP, LVL1, E. coli


miniprep

5mL of E.Coli containing the potential LvL1 Plasmid of the 3HP+GFP Sensor were centrifuged and the supernatant discarded.

For the Extraction ThermoScientific GeneJet Miniprep Kit was used and the protocol applied with slight variation

The filter was eluated in two steps 35µL of ddWater for 5min and another 15µL of ddH2O for 5min and the Eppi centrifuged in between eluation steps

c(3HP-GFP): 214.2 ng/µL

digest

6.5µL ddH2O

1.0µL 10x CutSmart Buffer NEB

1.0µL potential 3HP+GFP

0.5µL pst1 restriction enzyme

0.5µL eco1 restriction enzyme

incubated at 37°C for 1.5h

gel

1% agarose, 2µL Midori Green on 100mL Gel

run-time: 1:15h

fragment size as predicted

sequencing

2µL Primer (oiGEM0004_rev) 2µL Plasmid 8µL ddH2O

3HPGFP1.jpg


created: 17.07.2018 11:10updated: 19.07.2018 15:42

Author: Franziska NouschEntry 15/42: Transforamtion LvL1 potential 3HP-GFP Sensor in E.ColiIn Project: Team SensorWith tags: Sensor, 3HP, LVL1, E. coli

1. Thaw Aliquots (NEB)

2. Insert Plasmid (3HP-GFP: 1µL, PYTK: 1µL)

3. Leave on ice for 30min

4. Heatshock (42°C 60s)

5. Incubated on ice for 5min

6. Incubated at 37°C 200rpm for 90min

7. Plated (3HP-GFP on KAN, PYTK on Cm)

8. incubated at 37°C over night

18.7.19

many colonies on control and 3HP-GFP

three picked and incubated in liquid culture a' 5mL with KAN 20µg/mL

19.7.19

Vials were centrifuged for three separate cryostocks

the supernatant was discarded

cell pellets resuspended in 250µL LBI

250µL 40% glycerol was added

frozen in liquid nitrogen and stored in the -78° freezer

NEB§HPGFP.jpg


NEB3HPUV.jpg


created: 19.07.2018 14:51updated: 24.07.2018 18:49

Author: Franziska NouschEntry 17/42: Electroporation of V. natriegens with 3HP-GFP LVL1In Project: Team SensorWith tags: 3HP, LVL1, Sensor, V. natriegens, electrocompetent, electroporation

preparation of BHI V2 recovery Media

conc 1L 100mL 50mL

BHI 37 3.7 1.85

NaCl 58.44 204mM 11.92 1.192 0.596

KCl 74.55 4.2mM 0.313 0.0313 0.01565

MgCl2 (x6H2O)

203.3 23.14mM

4.7 0.47 0.235

Sucrose 342.3 680mM 232.76 23.276 11.638

Substances were added to a Falcon Tube and filtered (sterile)

aliquots of 500µL were made for storage.

one was stored at 37°C for later use.

recovery medium = BHI-rec.

Electroporation with LVL1 3HP-GFP-Sensor

One Aliquot of V.natriegens was thawed on ice

the LVL1 plasmid 3HP-GFP & the cuvette was chilled to 0°C

the plasmid was added to the cells (100ng) and put into the cuvette and electroporated at 700V, 25µF and 200Ohm

immediately afterwards 250µL of preheated recovery media (37°C) was added.

The cells were retrieved from the cuvette and placed in additional 250µL of BHI-rec. (total of 500µL)

The cuvette was flushed several times to account for maximum recovery.

The cells were incubated at 37°C at 200rpm for 2h.

3HP-GFP_Vn.jpg


They were plated on preheated (37°C) LBII KAN200 plates provided by D.Marchal and incubated at 37°C

23.07.18

many colonies on the plates. Digestion Pending.

3 colonies were singled out and grown in liquid LB Media for Cryo Stock and Digestion.


created: 24.07.2018 09:17updated: 24.07.2018 09:41

Author: Benjamin DanielEntry 19/42: Level 1 Assembly of the RFP and Lux 3HP sensorsIn Project: Team SensorWith tags: 3-HP, 3HP, 3HP Sensor, level 1, Sensor, transformation

After the successful assembly of the GFP sensor, the same approach was repeated for the RFP and Lux variants of the 3-HP sensor.

Time Temperature in °C

1 2 min 37

2 5 min 16

Goto 1, repeat 60 times

3 60 min 60

4 10 min 80

5 Hold 4

The resulting ligation was then used to transform chemically competent NEB turbo E. coli.

After regeneration in SOC media at 37°C for 1.5 hours, the cells were centrifuged gently at 4,500 rpm for 2 minutes,

the supernatant was removed by tipping the tubes and then the pellet was resuspended in the remaining medium.

This was plated on Kanamycin LB plates and incubated at 37°C over night.

image.png


created: 24.07.2018 10:16updated: 24.07.2018 10:24

Author: Benjamin DanielEntry 20/42: Creating a cellfree expression system to test the 3HP GFP sensorIn Project: Team SensorWith tags: BL21, 3-HP, 3HP Sensor, E. coli, level 1, level 1, level 1, level 1, Sensor, transformation

In order to create a cell free expression system, E. coli cells of the strain BL21 are transformed with the plasmid carrying the genes for the 3 HP sensor.

The strain BL21 had all proteases deleted, so there wil be no degradation in the cell free system, increasing its longevity.

The BL21 cells are chemically competent and transformed using the usual protocol.

After transformation, the cells recover for 1.5 hours at 37°C and are then plated on LB Kan plates.

After overnight incubation at 37°C, a single colony is picked and inoculated in a of LB Kan media.bigger volume


created: 24.07.2018 14:51updated: 01.10.2018 10:35

Author: Benjamin DanielEntry 21/42: No entry title yetIn Project: Team SensorNo tags associated


created: 25.07.2018 13:54updated: 25.07.2018 17:03

Author: Franziska NouschEntry 22/42: No entry title yetIn Project: Team SensorNo tags associated

miniprep

Four overnight culture 4mL of Vn containing the potential LvL1 Plasmid of the 3HP+GFP Sensor were centrifuged and the supernatant discarded.

For the Extraction ThermoScientific GeneJet Miniprep Kit was used and the protocol applied.

The filter was eluated in two steps 30µL of ddWater for 5min and another 20µL of ddH2O for 5min and the Eppi centrifuged in between eluation steps

Vn1: c(3HP-GFP): 440 ng/µL




digest for Vn1 and Vn3

Vn1 component

Vn2

0.5 DNA 1.5

0.5 EcoR1 0.5

0.5 Pst1 0.5

1.0 NEB CutSMART Buffer

1.0

7.5 H2O 6.5

incubated for ~1.5h

loaded on 1% agarose with midori green

runtime ~30min

Vn-3HP-digest.jpg


created: 27.07.2018 13:24updated: 27.07.2018 14:02

Author: Benjamin DanielEntry 23/42: First Platereader experiment featuring the 3HP GFP Sensor piGEM2505In Project: Team SensorWith tags: 3-HP, 3HP, 3HP Sensor, Sensor, frensh press

For testing the newly created Level 1 plasmid, carrying the 3HP sensor with sfGFP as reporter, we performed a platereader experiment.

Because we are not sure about the membrane permeability of 3-HP, also a crude cell lysate was used in the experiment.

1. Cell lysate

An overnight culture of V. natriegens containing the piGEM2505 plasmid was used to inoculate 600 mL of LBv2.

The culture was grown to an OD600 of 1 and then harvested by centrifuging it at 5.000 r.p.m. for 15 minutes in a centrifuge at 4°C.

The supernatant was discarded and the resulting pellet resuspended in 40 mL LBv2. This was passed through the frensh press three times at about 1,000 p.s.i..

The lysate was then cleared of cell debris by centrifuging it at 18,000 r.p.m. for 30 minutes at 4°C.

This lysate was then pipetted in the cool room into the wellplate and mixed with the appropriate amount of 3-HP to create the desired concentration.

2. Overnight culture of V. natriegens

An Overnight culture of V. natriegens was used to inoculate 20 mL of LBv2 media.

This was grown to an OD600 of 0.5 and then pipetted into the well plate in the cool room into the wellplate and mixed with the appropriate amount of 3-HP to create the desired concentration.

The resulting curve was inconclusive. There may have been a tecnical problem with the plate reader where you can see the stark drop offs.

Layout of the well plate:


1 2 3 4 5 6 7 8 9 10 11 12

A Lysate W/O 3HP

Lysate + .01 mM

Lysate +.04 mM

Lysate +.08 mM

Lysate +.16 mM

Lysate +.32 mM

Lysate +.64 mM

Lysate +1.3 mM

Lysate +2.5 mM

Lysate +5 mM

Lysate +10 mM

Lysate +20 mM

B Lysate W/O 3HP

Lysate W/O 3HP

Lysate W/O 3HP

Lysate W/O 3HP

Lysate W/O 3HP

Lysate W/O 3HP

Lysate W/O 3HP

Lysate W/O 3HP

Lysate W/O 3HP

Lysate W/O 3HP

Lysate W/O 3HP

Lysate W/O 3HP

C Culture W/O 3HP

Culture + .01 mM

Culture +.04 mM

Culture +.08 mM

Culture +.16 mM

Culture +.32 mM

Culture +.64 mM

Culture +1.3 mM

Culture +2.5 mM

Culture +5 mM

Culture +10 mM

Culture +20 mM

D Culture W/O 3HP

Culture W/O 3HP

Culture W/O 3HP

Culture W/O 3HP

Culture W/O 3HP

Culture W/O 3HP

Culture W/O 3HP

Culture W/O 3HP

Culture W/O 3HP

Culture W/O 3HP

Culture W/O 3HP

Culture W/O 3HP

E

F

G

H

Lysate_chart.JPG


V._natriegens_chart.JPG


created: 27.07.2018 14:06updated: 27.07.2018 14:11

Author: Benjamin DanielEntry 24/42: Creation of chemocompetent DL21 E.coli cellsIn Project: Team SensorWith tags: E. coli, chemocompetent, DL21

Day 1: Innoculate the cells in 10ml LB Medium and Grow Overnight.

Day 2: From the O/N culture Freshly innoculate 0.1 - 0.5% of total volume LB, e.g. 0.5 mL for 200mL.

Innoculum into 100ml of LB Medium and start growing the cells freshly till the O.D of the Culture reaches 0.4 - 0.5 at A600nm.

1) Once the O.D was reached incubate the cultore at 4 degrees for 30 minutes

2) Take the culture into two 50ml centrifuge tubes and spin down the cells ( Maintain Centrifuge at 4 degrees) ( 5000rpm - 5mins)

3) Discard the sup and resuspend the cells in 1/2 the original Volume (25 ml) of ice Cold 0.1M MgCl2 and and incubate in ICE for 15 mins4)

Centrifuge the cells and discard the sup ( 5000rpm - 5mins)

5) Again resuspend the cells in 1/2 the original Volume (25 ml) of ice Cold 0.1M CaCl2 and and incubate in ICE for 15 minutes

6) Centrifuge the cells and discard the sup ( 5000rpm - 5mins)

7) Resuspend the cells in 1ml of ice Cold 0.1M CaCl2+20% Glycerol and aliqoute 100ul into each 1.5 ml tubes and store them immediately in -70 degrees.


created: 31.07.2018 17:41updated: 03.08.2018 10:07

Author: Benjamin DanielEntry 25/42: Comprehensive Test of the 3 HP GFP Sensor in three different strainsIn Project: Team SensorWith tags: 3HP, 3HP, 3HP Sensor, DL21, V. natriegens, NEB Turbo, platereader

0.01 mM 0.04 mM 0.08 mM 0.16 mM 0.32 mM 0.64 mM 1.3 mM 2.5 mM 5 mM 10 mM

1 2 3 4 5 6 7 8 9 10

A NEB WT NEB WT NEB WT NEB WT NEB WT NEB WT NEB WT NEB WT NEB WT NEB WT

B NEB 3HP NEB 3HP NEB 3HP NEB 3HP NEB 3HP NEB 3HP NEB 3HP NEB 3HP NEB 3HP NEB 3HP

C DL21 WT DL21 WT DL21 WT DL21 WT DL21 WT DL21 WT DL21 WT DL21 WT DL21 WT DL21 WT

D DL21 3HP DL21 3HP DL21 3HP DL21 3HP DL21 3HP DL21 3HP DL21 3HP DL21 3HP DL21 3HP DL21 3HP

E V. natriegens WT

V. natriegens WT

V. natriegens WT

V. natriegens WT

V. natriegens WT

V. natriegens WT

V. natriegens WT

V. natriegens WT

V. natriegens WT

V. natriegens WT

F V. natriegens 3HP

V. natriegens 3HP

V. natriegens 3HP

V. natriegens 3HP

V. natriegens 3HP

V. natriegens 3HP

V. natriegens 3HP

V. natriegens 3HP

V. natriegens 3HP

V. natriegens 3HP

G LB LB 3HP LB Kan empty empty empty empty empty empty empty

H LBv2 LBv2 3HP LBv2 Kan empty empty empty empty empty empty empty

Unbenannt.JPG


created: 01.08.2018 13:40updated: 02.08.2018 08:43

Author: Franziska NouschEntry 26/42: Golden Gate (NEB) Assembly of 3HP-RFP and 3HP-LUXIn Project: Team SensorNo tags associated

Electroporation with LVL1 3HP-RFP-Sensor (and 3HP-LUX)

One Aliquot of V.natriegens was thawed on ice

the LVL1 plasmid 3HP-RFP (LUX) the cuvette was chilled to 0°C

the Golden Gate was (10µL) added to the cells and put into the cuvette and electroporated at 700V, 25µF and 200Ohm

immediately afterwards 110µL of preheated recovery media (37°C) was added.

The cells were retrieved from the cuvette and placed in additional BHI-rec. (total of 500µL)

The cuvette was flushed several times to account for maximum recovery.

The cells were incubated at 37°C at 200rpm for 1.5h.

They were plated on preheated (37°C) LBII KAN200 plates and incubated at 37°C

No cells were visible after24h.


created: 15.08.2018 09:17updated: 15.08.2018 09:30

Author: Benjamin DanielEntry 27/42: Preliminary testing of the LUX 3HP BiosensorIn Project: Team SensorWith tags: 3-HP, 3HP Sensor, platereader, V. natriegens, Sensor, LUX

In order to get a quick idea of weather the 3HP sensor with LUX as reporter had enough sensitivity to show induction by 3HP, a crude experiment was performed.

Two overnight cultures of V. natriegens, expressing the 3HP sensor, were induced by addition of 5mM 3HP and subsequent incubation at 37°C for 3 hours. Then, the cultures were placed in to a 96 well plate, each culture in two separate wells.

As controls, uninduced cultures from the same overnight culture, WT V. natriegens and LBv2 media was used.

The measured fluorescence indicated a 797-27 fold induction of the LUX expression over the background.

LUX_Bauer_1+2_5mM_3HP.JPG


created: 20.08.2018 10:17updated: 20.08.2018 10:31

Author: Benjamin DanielEntry 28/42: Double Transformation of two Plasmids into V. natriegensIn Project: Team SensorWith tags: electrocompetent, transformation, PYTK, LUX, Sensor, V. natriegens

In order to test, weather a cotransformation of two Plasmids into V. natriegens was possible via electroporation, we used the two plasmids PYTK and Lux 3-HP sensor Lvl1 and inserted them into WT V. natriegens by means of electroporation.

The PYTK plasmid is about 3.5 kB in size and the Lux 3-HP sensor Lvl1 about 10 kB. PYTK carries a chloramphenicol resistance and a expresses GFP constitutively, while the Lux 3-HP sensor Lvl1 plasmid carries a kanamycin resistance and expresses the Lux 3-HP sensor.

After elecctroporation, the cells were recovered for 1.5 h at 37° C and then plated on LB2 kanamycin ( 200µg/mL) plates and incubated overnight at 37°C.

On the following morning, about 60 colonies were visible on the plate, of which about 30 also expressed GFP.

These were restreaked on combined kanamycin ( 200µg/mL) and chloramphenicol (2 µg/mL) and again incubated at 37°C for two days. The result were colonies that expressed GFP while being able to grow.

This indicates, that the antibiotics forced V. natrigens to propagate both plasmids.

20180820_095624.jpg


created: 22.08.2018 14:22updated: 22.08.2018 14:28

Author: Benjamin DanielEntry 29/42: Amplification of FapO from B. subtilisIn Project: Team SensorWith tags: fapO, PCR, B.subtilis, Malonyl-CoA, Sensor


In Order to amplify fapO from the B. subtilis 168 genome, the following settings and PCR mix were used.

Total: 50 µL

PCR 2x Phanta Buffer 25 µL


2 µL

dNTPs 1 µL

Genomic DNA B. subtilis 168

1.5 µL

Phanta Max Phusion Super Polymerase

1 µL


2 µL

H 02 17.5 µL



95°C 30 s

2. denaturation

95°C 15 s



5. final extension

72°C 90 s



created: 23.08.2018 10:42updated: 26.09.2018 15:55

Author: Benjamin DanielEntry 30/42: Characterization of the 3-HP sensor in V. natriegens and E. coliIn Project: Team SensorWith tags: 3HP Sensor, 3HP, 3-HP, E. coli, Sensor, LUX, V. natriegens, NEB Turbo, e.coli

In order to see how well the 3-HP sensor performs using different reporter genes, organisms and 3-HP concentrations, E. coli and V natriegens strains expressing all combinations were grown overnight. In the morning their OD was normalized to 0.05 and incubated for 5 hours with different concentrations of 3-HP. They were then transferred into a 96 well plate and all parameters were measured.

Messung.JPG


created: 23.08.2018 11:20updated: 23.08.2018 11:24

Author: Benjamin DanielEntry 31/42: Check for the right size of the amplification for FapRIn Project: Team SensorWith tags: B.subtilis, fapR, Fap, E. coli, PCR, Sensor, Malonyl-CoA

20180823_110339.jpg

The expected size for the band of the right amplification product fot fapR is 567bp the observed bands are somewhat above the 500bp marker, therefor it is concludet, that the amplification was successful.


created: 24.08.2018 14:04updated: 24.08.2018 14:04

Author: Benjamin DanielEntry 32/42: Level 0 Assembly of fapRIn Project: Team SensorWith tags: B.subtilis, Fap, level 0, fapR, Sensor, transformation, E. coli, chemokompetent, Malonyl-CoA


created: 30.08.2018 15:41updated: 30.08.2018 15:44

Author: Benjamin DanielEntry 33/42: Creation of chemocopmetent NEB Turbo E.coliIn Project: Team SensorWith tags: chemocompetent, NEB Turbo, E. coli

Materials

TB buffer

10 mM HEPES pH 6.715 mM CaCl255 mM MnCl2250 mM KCl

Mix all component except MnCl and adjust the pH to 6.7 with KOH. Then add the MnCl and filter sterilize the mixture using a 0.22-2 2µm filter.

LB medium:

20 g/L bacto-tryptone5 g/L yeast extract5 g/L NaCl

SOB medium++

20 g/L bacto-tryptone5 g/L yeast extract0.5 g/L NaCl0.186 g/L KCl10 mM MgCl210 mM MgSO4

Heat sterilize the medium and add sterile MgCl and MgSO just before use.2 4

Stock solutions:

1 M MgCl (sterilized)21 M MgSO (sterilized)4DMSO

Procedure


1. Inoculate 3 ml LB medium with the appropriate strain and incubate the culture overnight at 37°C.E. coli

2. Add the overnight culture to 500 ml SOB++ medium and incubate the culture at 25-30°C until the absorbance at 600 nm was approx. 0.5 (between 0.4 and 0.6).In the original paper the culture was incubated at 18°C but in our hands this did not make a difference. Good competent cells were also obtained when LB or SOC medium was used.

3. Chill the culture for at least 10 min on ice.In the following steps, the cell suspension should be kept on ice as much as possible.

4. Centrifuge the cell suspension for 10 min at 4,500 rpm (Sorvall RCB4 rotor) or 6,000 rpm (Sorvall GSA rotor) at 4°C.

5. Gently resuspend the pellet in 100 ml ice-cold TB buffer.

6. Incubate the cell suspension on ice for 10 min.

7. Centrifuge for 10 min at 4000 rpm (Sorvall SS-34 rotor) at 4°C.

8. Gently resuspend the pellet in 18.6 ml ice-cold TB buffer and add 1.4 ml DMSO.

9. Incubate the cell suspension on wet ice for at least 10 min.

10. Aliquot the cell suspension at 600 µl per tube.

11. Shock-freeze the cell suspension in liquid nitrogen and store the tubes at -80° or in liquid nitrogen.At -80°C the cells will be competent for at least 6 months. In liquid nitrogen they will stay competent indefinitely.

Remarks:

The transformation efficiency of the competent cells can be determined using 5 ng of pure plasmid DNAand 200 µl competent cell suspension (see transformation protocol).

This will give approx. 1 x 10 transformants per µg plasmid DNA for DH5a.8

Reference

Inoue . (1990) , 23-28.et al Gene 96


created: 08.09.2018 13:18updated: 08.09.2018 13:32

Author: Benjamin DanielEntry 34/42: Test PCR of Lvl2 MalonylCoA sensorsIn Project: Team SensorWith tags: fapR, Fap, fapO, E. coli, e.coli, Sensor, Malonyl-CoA, PCR

Unbenannt.JPG


To identify the correctly assembled Level 2 parts, a test PCR was performed on the purified plasmid from the colonies of the MaCoA sensor.

For the RFP sensor, oiGEM1007 and 06_GA_PYTK were used. For the GFP sensor, oiGEM1017 and 07_GA_pYTK.

Total: 5 µL

Primer forward: 0.5 µL

dNTPs 0.5 µL

Template 0.25 µL

Phusion Polymerase

0.5 µL


0.5 µL

2.25 µL



95°C 30 s

2. denaturation

95°C 15 s



5. final extension

72°C 90 s


The gel showed the expected bands at around 500-600 bp.


created: 10.09.2018 14:14updated: 10.09.2018 14:25

Author: Benjamin DanielEntry 35/42: Test PCR of Lvl2 MalonylCoA sensorsIn Project: Team SensorWith tags: 3HP Sensor, 3-HP, fapO, Fap, fapR, PCR, LUX, E. coli, Sensor, 3HP

Test PCR for Malonyl CoA sensors. The expected bands are 1300 bp for RFP, 1400 for GFP and 2900 for LUX.

Total: 5 µL

Primer forward: 0.5 µL

dNTPs 0.5 µL

Template 0.25 µL

Phusion Polymerase

0.5 µL


0.5 µL

2.25 µL



95°C 30 s

2. denaturation

95°C 15 s



5. final extension

72°C 90 s



Unbenannt.JPG


created: 18.09.2018 11:14updated: 20.09.2018 17:16

Author: Franziska NouschEntry 36/42: No entry title yetIn Project: Team SensorNo tags associated


Biosensor-response-test

The Biosensors, MaCoA-RFP & MaCoA-GFP & 3HP-SSR-Lux where tested with the respective concentration of Malonyl-CoA and 3HP

The response was measured in a black 96-well plate with the given concentration in the table at 37°C while shaking. An over-night culture holding the respective plasmid was used to inoculate a fresh culture and grown to an OD600 between 0.3 and 0.6 before measurement. The plate was stored at 10°C beforehand. V = 200µL.well

Experiment set-up was derived from

Unfortunately, no significant signals could be detected. Since no response could be detected for LUX, the whole 3HP-SSR-Lux Plasmid might be flawed. Strangely the 3HP-SSR-Lux displayed fluorescence activity.

96-well-plate:

1 2 3 4 5 6 7 8 9 10 11 12

MaCoA GFP

A 0 2 4 8 10 12 14 16 18 20 22 25 µM

MaCoA GFP

B 0 2 4 8 10 12 14 16 18 20 22 25 µM

MaCoA RFP

C 0 2 4 8 10 12 14 16 18 20 22 25 µM

MaCoA RFP

D 0 2 4 8 10 12 14 16 18 20 22 25 µM

E M M MaCoA + H2O

MaCoA + H2O

MaCoA+M MaCoA+M 3HP + H2O

3HP + H2O

3HP+M 3HP+M

F

3HP-Lux G 0 4 8 16 32 64 125 250 500 µM

3HP-Lux H 0 4 8 16 32 64 125 250 500 µM

Comment: Analysis of the data revealed that the fluorescence of 3HP-Lux can be attributed to a lower OD and thereby higher media fluorescence.

140918_Sensor_Test_Fluo.xlsx

140918_Sensor_Test_Lux.xlsx

https://eln.labfolder.com/eln/workspace/block/file/2917865/3/140918_Sensor_Test_Fluo.xlsx

https://eln.labfolder.com/eln/workspace/block/file/2917865/4/140918_Sensor_Test_Lux.xlsx


140918_Sensor_Test_OD.xlsx

https://eln.labfolder.com/eln/workspace/block/file/2917865/5/140918_Sensor_Test_OD.xlsx


created: 28.09.2018 15:05updated: 28.09.2018 15:14

Author: Benjamin DanielEntry 37/42: Ligating the ALD into the pJET 1.2 VectorIn Project: Team SensorWith tags: ligation, pJET 1.2, E. coli

For the ligation of the synthesized ALD gene into the pJET1.2 verctor, the following protocol was used.

1µL T7 Ligase

1µL T7 Buffer

1µL ALD Synthese (10 ng/µL)

2µL pJET 1.2

5µL H2O

The ligation was incubated at room temperature for 3 hours, then transformed into chemocompetent E. coli. These were plated on Ampicillin LBI plates and incubated overnight.

Then single colonies were inoculated in liquid media and grown to medium high cell density. The plasmid was extracted from the cells using the GeneJet Plasmid extraction kit from Thermofisher.

Then the plasmids were run on a gel to check for the presence of the inserted ALD. Then the plasmids were send for sequencing.


created: 01.10.2018 10:35updated: 01.10.2018 10:52

Author: Benjamin DanielEntry 40/42: Finding the problem with theAmplification of the 3-HP sensor from P. putida genomic DNAIn Project: Team SensorWith tags: 3-HP, 3HP, 3HP Sensor, Genomic DNA, Pseudomonas putida, Sensor, PCR


Since the amplification of the 3-HP sensor did not seem to function, while it previously clearly did, the PCR parameters were varied to find possible sources for the problem.

All four variations ran on the same thermocycler, with the same thermocycler protocol. A new working stock of the primers was prepared as well as a new stock of buffer opened.

95°C 300s

95°C 30s

58°C 30s

72°C 180s

Go to: 2, repeat 30X

4°C hold

The reaction mixtures were prepared as follows:

iteration 1 2 3 4

H2O 37.5µL 35.5µL 38.5µL 20µL

Buffer 10x 5µL 5µL 5µL 25µL Phanta Buffer

template 2µL 1µL 1µL 1µL

P1 1µL 2µL 1µL 1µL

P2 1µL 2µL 1µL 1µL

dNTPs 1µL 1µL 1µL 1µL

Polymerase 1µL Bange Concoction

0.5µL Bange Concoction

1µL Bange Concoction

1µL Phanta Max

DMSO 1.5µL 1.5µL 1.5µL -

Since the PCR without the Bange Concoction Phusion mix worked, I suggest for future PCRs not to rely on that.


20181001_102030.jpg


created: 05.10.2018 11:44updated: 05.10.2018 11:48

Author: Benjamin DanielEntry 41/42: Screening for correct LVL 0 3-HP SensorIn Project: Team SensorWith tags: E. coli, PCR, LUX, 3HP, 3-HP, 3HP Sensor, Sensor

In order to determine which of the non fluorescent colonies of the LVL0 assembly transformation were correct, first a colonie PCR was performed using the ONETAQ polymerase from NEB. For the right insert, the band should have a size of 1120 bp.

Most colonies harbor the right insert. The are grown and send for sequencing.

Colony_PCR_mit_beschriftung.png


created: 10.10.2018 18:52updated: 11.10.2018 10:31

Author: Benjamin DanielEntry 42/42: Erit LuxIn Project: Team SensorWith tags: 3-HP, 3HP, 3HP Sensor, E. coli, e.coli, NEB Turbo, LUX, Sensor, transformation, Pseudomonas putida

E. coli cells expressing lux. They are Level 1 constructs, as described before.

Felix_Lux.Tif

In Project: Team Sensor With tags: fapR, Malonyl-CoA ...2018.igem.org/wiki/images/3/32/T--Marburg... · Project: Team Sensor Page 5 Step Temperature Time 1. initial denaturation 98°C

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