Difference between revisions of "Team:Aalto-Helsinki/InterLab"

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<h3>Cultivations and measurement</h3>
 
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<p>Followed the next protocol for preparing the samples:</br></br>Streaked out LB-plates with E.coli TOP10 strain containing every device and control with chloramphenicol concentration of 35 ug/ml. Incubated plates overnight (18-20 hours) at 37\(^\circ\)C. Picked up biological triplates from the plates and inoculated 3 ml liquid cultures from the colonies with 12 ml polypropylene test tube. Incubated the tubes at 37\(^\circ\)C with shaking at 300rpm for 18 hours. Measured OD600 values of cultures in cuvettes and calculated the dilution required for each sample to obtain the OD value of 0.5. Diluted and re-measured the samples to reach 5% accuracy of 0.5. Transfered 200ul of each sample into 96-well transparent plate and set the platereader to read fluorescence intensity. Proceed with the measurements with the following equipment:  </br> </br>Type: Cell Imaging Multi-Mode Plate Reader </br>
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<p>Followed the next protocol for preparing the samples:</br></br>Streaked out LB-plates with E.coli TOP10 strain containing every device and control with chloramphenicol concentration of 35 &#956;g/ml. Incubated plates overnight (18-20 hours) at 37\(^\circ\)C. Picked up biological triplates from the plates and inoculated 3 ml liquid cultures from the colonies with 12 ml polypropylene test tube. Incubated the tubes at 37\(^\circ\)C with shaking at 300rpm for 18 hours. Measured OD600 values of cultures in cuvettes and calculated the dilution required for each sample to obtain the OD value of 0.5. Diluted and re-measured the samples to reach 5% accuracy of 0.5. Transfered 200 &#956;l of each sample into 96-well transparent plate and set the platereader to read fluorescence intensity. Proceed with the measurements with the following equipment:  </br> </br>Type: Cell Imaging Multi-Mode Plate Reader </br>
 
Name and model: Cytation 3 </br>
 
Name and model: Cytation 3 </br>
 
Company: BioTek Instruments, Inc</br></br>
 
Company: BioTek Instruments, Inc</br></br>

Revision as of 21:16, 12 September 2015

InterLab Study

In the Interlab study, iGEM teams gather fluorescence data for three GFP expressing devices around the world using the same methods for cloning and measurements. Our InterLab Study Lab Book can be found here for a fully detailed overview of our study.

Devices

The following devices were created in a backbone pSB1C3:

⚫ J23101 + I13504 (B0034-E0040-B0015)
⚫ J23106 + I13504 (B0034-E0040-B0015).

However, constructing a device J23117 + I13504 (B0034-E0040-B0015) wasn't successful due to the time limit and limited ligation times. More details available on Lab Book. The device sizes were analyzed with restriction mapping which results can be seen in Figure 1.

Used BBa_I20270, a GFP expressing part in the pSB1C3 backbone as a positive control. Negative controls were organisms with an empty plasmid and without any vector.

Protocols

Constructing devices

Protocols for the restriction, ligation and transformation of BioBricks can be found here.

Cultivations and measurement

Followed the next protocol for preparing the samples:

Streaked out LB-plates with E.coli TOP10 strain containing every device and control with chloramphenicol concentration of 35 μg/ml. Incubated plates overnight (18-20 hours) at 37\(^\circ\)C. Picked up biological triplates from the plates and inoculated 3 ml liquid cultures from the colonies with 12 ml polypropylene test tube. Incubated the tubes at 37\(^\circ\)C with shaking at 300rpm for 18 hours. Measured OD600 values of cultures in cuvettes and calculated the dilution required for each sample to obtain the OD value of 0.5. Diluted and re-measured the samples to reach 5% accuracy of 0.5. Transfered 200 μl of each sample into 96-well transparent plate and set the platereader to read fluorescence intensity. Proceed with the measurements with the following equipment:

Type: Cell Imaging Multi-Mode Plate Reader
Name and model: Cytation 3
Company: BioTek Instruments, Inc

The following parametres were used:

Excitation wavelength: 470 nm
Emission wavelength: 511 nm
Optics Position: Bottom
Filter: Quadruple Monochromators with Bandwith of 16 nm
Units Reported: RFU

Results

The measurements were partially successful. Results can be seen in tables below. The data hasn't been processed further and the units are in Relative Fluorescence Units.

Most samples generated overflow values which could not be measured with the plate reader. The incubation may have generated too much fluorescence proteins even though the samples were diluted properly. The problem wasn't solved after new dilutions of 9:10 ratio and due to the time limit, other ratios weren't attempted. The first blank value is with antibiotics and the second without for the negative control without any plasmids.

1st Technical Replicon

Sample

J23117 + I13504

J23101 + I13504

J23151

Empty backbone

Without backbone

Blanks

1

OVRFLW

83954

53925

16309

13663

29336

2

OVRFLW

OVRFLW

56764

16335

14148

14940

3

OVRFLW

OVRFLW

61918

16771

14073

2nd Technical Replicon

Sample

J23117 + I13504

J23101 + I13504

J23151

Empty backbone

Without backbone

Blanks

1

OVRFLW

85224

53553

16307

13747

29470

2

OVRFLW

OVRFLW

56472

16204

14267

14972

3

OVRFLW

OVRFLW

60969

16818

14381

3rd Technical Replicon

Sample

J23117 + I13504

J23101 + I13504

J23151

Empty backbone

Without backbone

Blanks

1

OVRFLW

86750

56390

16123

13536

30778

2

OVRFLW

OVRFLW

58294

16294

14207

14856

3

OVRFLW

OVRFLW

62229

16793

13994

Restriction Map

The gel electrophoresis mapping of J23101 + I13504 and J23106 + I13504 can be seen in Figure 1.

Figure 1. Restriction mapping for J23101 + I13504 and J23106 + I13504.

Comments are missing because the figure will be changed later . The sizes of samples seems to be slightly bigger than expected so a new restriction and gel run will be proceeded during the week 37. The explanations for ladders and scaling will be updated then.