Background

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Design

Materials and methods

Used plasmids

1. Device 1 (20K+21J): J23101 + I13504, in the pSB1C3 backbone

2. Device 2 (22A+21J): J23106 + I13504, in the pSB1C3 backbone

3. Device 3 (22K+21J): J23117 + I13504, in the pSB1C3 backbone

4. Positive control (C+): I20270, in the pSB1C3 backbone

5. Negative control (C-): K823008, in the pSB1C3 backbone

Used strain

E. coli E5alpha

Used material

1. LB-M agar plates with chloramphenicol

2. LB-M agar plates with ampicillin

3. 1,5 ml eppendorf tubes

4. 0,5 ml PCR tubes

5. 50 ml centrifuge conical base and rim tubes

6. NucleoSpin Plasmid DNA, RNA, and protein purification Kit

7. NucleoSpin Gel and PCR Clean-up Kit

8. LB-M medium with chloramphenicol

9. NaOH agarose gel and buffer

10. Sphero Rainbow Calibration Particles, 8 Peaks, 3.0-3.4

Used methods (links)

1. Transformation

2. Miniprep

3. Restriction digest

4. Ligation

5. NucleoSpin Gel Clean-up

6. NucleoSpin Plasmid DNA purification

7. Flow cytometry

Used machines

1. Incubator: Binder, ATP.line CB(E6), CO2 – Incubator with O2 control, model CB160, 230V

2. Orbita Shaker PSU-10i, 280 rpm, 360deg

3. Flow Cytometr C6

4. Thermo-Shaker with cooling for microtubes and PCR plates TS-100C

5. Centrifuge 5424/5424 R

Used software

1. CFlow Plus

2. Microsoft Excel

3. Sphero PMT QC Template

Description

Plasmids containing promoters and GFP were taken from The 2015 DNA Distribution Kit and transformed into E. coli. Colonies were streaked for patches and amplified plasmids were obtained using NucleoSpin Plasmid DNA purification. Restriction digest followed using XbaI and PstI enzymes for digest of plasmid with GFP and SpeI and PstI ezymes for digest of plasmids containing promoters. Desired parts were then cut out of gel and purified using NucleoSpin Gel Clean-up. Ligation of Device 1 and 2 followed our standard protocol. However ligation of Device 3 had to be repeated several times and the protocol had to be modified (2 hours ligation, 5:1 insert:vector ratio). All devices were transformed into E.coli. Fluorescence of colonies was checked up under UV light. Plasmids were then obtained by NucleoSpin Plasmid DNA purification. Devices were verified on gel after restriction digest using SpeI and EcoRI enzymes and by sequencing.

Picture o patches (all together)

Picture of colonies????

All verified devices were again transformed into E. coli accompanied by positive and negative control. 5 ml of liquid LB-M medium with chloramphenicol were inoculated with three chosen colonies of each device. There were no biological replicates for positive and negative control. Liquid cultures were incubated for 17 hours in Orbita Shaker PSU-10i placed in incubator. OD of these cultures was measured and diluted to 0,1. Fluorescence of biological and also technical replicates was measured using Flow Cytometr C6 following our flow cytometry protocol.

Protocols

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