2018 InterLab: Form III

Form III: Cell Measurements

Every team that participates in the 2018 iGEM InterLab study needs to fill in three forms. This is Form III.

This form is part of your InterLab submission, and will ask you questions about your cell growth protocol, as well as the protocol you used for your fluorescence measurements in your cells. Fill this in as best as you can. Use of E. coli K-12 DH5-alpha strain for this protocol is required.

This form follows the InterLab Plate Reader Protocol, pages 9-12 from this URL:

http://2018.igem.org/wiki/images/0/09/2018_InterLab_Plate_Reader_Protocol.pdf

The purpose of this form is to document your performance of the protocol, and to note any deviations from the protocol you may have made. This form will not provide a detailed protocol, so you must refer to the link above in order to perform these experiments.

If you have any questions or problems filling in this form, please email us at measurement at igem dot org.

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* 1. Team Name

Please enter your team name below. Please make sure it's the same as your official team name, including any hyphens or other characters.

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Workflow Overview

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* 2. Day 1: Transformation

Transform Escherichia coli DH5α with these following plasmids (each plasmid in a different sample). Please check off each device that you transformed. All devices are in the pSB1C3 backbone and confer chloramphenicol resistance. A detailed transformation protocol can be found here: http://parts.igem.org/Help:Protocols/Transformation

Negative control: BBa_R0040

Positive control: BBa_I20270

Test Device 1: BBa_J364000

Test Device 2: BBa_J364001

Test Device 3: BBa_J364002

Test Device 4: BBa_J364007

Test Device 5: BBa_J364008

Test Device 6: BBa_J364009

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* 3. Day 2: Cell Growth

Please check off each step that you followed. If you did anything differently or extra, please note that in the "Other" box.

Pick 2 colonies from each of plate and inoculate each in 5-10 mL LB medium + Chloramphenicol (For antibiotic concentrations, please follow these guidelines: http://parts.igem.org/Help:Protocols/Antibiotic_Stocks).

Grow the cells overnight (16-18 hours) at 37°C and 220 rpm.

With 8 total devices (including controls) and 2 colonies per device, you should have 16 cultures.

Other (please specify)

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* 4. Day 3 : Cell growth, sampling, and assay

For this part of the protocol, you will need to prepare two 96-well plates, one for each time point (0 hour and 6 hours). Please check off each step that you followed. If you did anything differently or extra, please note that in the "Other" box.

Set your instrument to read Abs600 (as OD calibration setting)

Measure Abs600 of the overnight cultures

Dilute the cultures to a target Abs600 of 0.02 in 12 ml LB medium + Chloramphenicol in 50 mL falcon tube.

Remove 500uL of each culture for your zero time point and hold these samples on ice. (You should have 16 sample tubes from the time=0 time point)

Incubate the cultures at 37°C and 220 rpm.

Remove 500uL samples of each culture after 6 hours of growth. You should have 16 sample tubes for each time point.

Set up your four measurement plates: For colony #1 culture, pipet 100 uL samples into wells A, B, C and D of the row for that device. For colony #2, pipet 100uL samples into wells E, F, G, and H of the row for that device.

Read your plates, taking care to use the exact same settings used for your fluorescein measurement.

Other (please specify)

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Recommended Plate Layout

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* 5. Measurement

It is important that you use the same instrument settings that you used when measuring the fluorescein standard curve. This includes using the same sample volume (e.g., 100 µl) for measurement using the spectrophotometer. Samples should be laid out according to the figure layout for Abs600 and Fluorescence measurement. Pipette 100 µl of each sample into each well. Set the instrument settings as those that gave the best results in your calibration curves (no measurements off scale). If necessary you can test more than one of the previously calibrated settings to get the best data (no measurements off scale). Please check off each step that you followed. If you did anything differently or extra, please note that in the "Other" box.

Measure OD and GFP fluorescence of all samples

Import data into blue cells in Excel (cell measurement) sheets provided

Other (please specify)

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* 6. Did you upload the Excel sheets to Dropbox?

Dropbox link can be found here: http://2018.igem.org/Measurement/InterLab/Plate_Reader

Make sure you name your Excel file following this template: 2018_InterLab_YourTeamName.xlsx

If you cannot access the Dropbox link, please email your completed Excel files to measurement AT igem DOT org.

Yes, it has been uploaded

No, we emailed it

Other (please specify)

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* 7. What type of media did you use for this step?

Standard E. coli growth media is LB (Luria Bertani) media, supplemented with the appropriate antibiotic (chloramphenicol for all devices). If you do not have access to LB, please enter detailed information about your media here. For antibiotic concentrations, please follow these guidelines: http://parts.igem.org/Help:Protocols/Antibiotic_Stocks

Luria Bertani

Other (please specify)

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* 8. What type of vessel or container did you use to grow your cells?

50 ml conical tube, amber or covered with foil (preferred)

Other (please specify)

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* 9. What temperature setting did you use during the measurement?

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* 10. What type of 96-well plates did you use?

black plates with transparent/clear bottom (preferred)

white plates with transparent/clear bottom

clear plates

Other (please specify)

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* 11. Did your plates have flat-bottomed or round-bottomed wells?

We strongly recommend teams to use flat-bottomed plates. This is of critical importance if you do a bottom reading with your plate reader.

Flat

Round

Other (please specify)

2018 InterLab: Form III

Form III: Cell Measurements

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* 1. Team Name

Please enter your team name below. Please make sure it's the same as your official team name, including any hyphens or other characters.

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Workflow Overview

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* 3. Day 2: Cell Growth

Please check off each step that you followed. If you did anything differently or extra, please note that in the "Other" box.

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* 4. Day 3 : Cell growth, sampling, and assay

For this part of the protocol, you will need to prepare two 96-well plates, one for each time point (0 hour and 6 hours). Please check off each step that you followed. If you did anything differently or extra, please note that in the "Other" box.

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Recommended Plate Layout

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* 8. What type of vessel or container did you use to grow your cells?

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* 9. What temperature setting did you use during the measurement?

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* 10. What type of 96-well plates did you use?

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* 11. Did your plates have flat-bottomed or round-bottomed wells?

We strongly recommend teams to use flat-bottomed plates. This is of critical importance if you do a bottom reading with your plate reader.

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* 12. Additional information

If you need more space to explain any adjustments to this form, please enter your comments in the box below.

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* 13. Feedback

Please let us know any other thoughts or comments you have about the InterLab study experience.

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* 1. Team NamePlease enter your team name below. Please make sure it's the same as your official team name, including any hyphens or other characters.

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Workflow Overview

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* 3. Day 2: Cell GrowthPlease check off each step that you followed. If you did anything differently or extra, please note that in the "Other" box.

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* 4. Day 3 : Cell growth, sampling, and assayFor this part of the protocol, you will need to prepare two 96-well plates, one for each time point (0 hour and 6 hours). Please check off each step that you followed. If you did anything differently or extra, please note that in the "Other" box.

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Recommended Plate Layout

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* 8. What type of vessel or container did you use to grow your cells?

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* 9. What temperature setting did you use during the measurement?

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* 10. What type of 96-well plates did you use?

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* 11. Did your plates have flat-bottomed or round-bottomed wells?We strongly recommend teams to use flat-bottomed plates. This is of critical importance if you do a bottom reading with your plate reader.

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* 12. Additional informationIf you need more space to explain any adjustments to this form, please enter your comments in the box below.

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* 13. FeedbackPlease let us know any other thoughts or comments you have about the InterLab study experience.

* 1. Team Name

Please enter your team name below. Please make sure it's the same as your official team name, including any hyphens or other characters.

* 3. Day 2: Cell Growth

Please check off each step that you followed. If you did anything differently or extra, please note that in the "Other" box.

* 4. Day 3 : Cell growth, sampling, and assay

For this part of the protocol, you will need to prepare two 96-well plates, one for each time point (0 hour and 6 hours). Please check off each step that you followed. If you did anything differently or extra, please note that in the "Other" box.

* 11. Did your plates have flat-bottomed or round-bottomed wells?

We strongly recommend teams to use flat-bottomed plates. This is of critical importance if you do a bottom reading with your plate reader.

* 12. Additional information

If you need more space to explain any adjustments to this form, please enter your comments in the box below.

* 13. Feedback

Please let us know any other thoughts or comments you have about the InterLab study experience.