Team:Shanghaitech/InterLab

233

Abstract

Repeatability is one of the most important part of biological experiment, however, it’s difficult to repeat the measurement data in different labs. As for the measurement of fluorescence, it can be influenced by various factors: bacterial strain, culture medium, plate reader and so on. Therefore, following a standard protocol strictly is one of the guarantee of the repeatable experimental conclusion. This year, we use plate reader rather than flow cytometry in 2016 (click here to view). Except for individual settings, we follow the standard protocol by iGEM authority completely, the data is reliable.

Plasmids and the prediction of results

All the eight plasmids are shown below:

From the table above, there are three different constitutive promotors (pcons) and two different ribosome binding sites (RBSs), which form six different combinations as Test Device 1-8.

(http://parts.igem.org/Part:BBa_J23101)

According to the data of iGEM parts, we know that the strength of these three pcons: J23101＞J23106＞J23117. However, there lacks the reliable data of the strength of pcon (J23151) and the comparison of three RBSs (B0032, B0034, J364100). In conclusion, we predict the result of the Interlab fluorescence measurement:

Test Device 1 ＞ Test Device 2 ＞ Test Device 3

Test Device 4 ＞ Test Device 5 ＞ Test Device 6

All the plasmid sequences (.dna file) can be download from: https://drive.google.com/file/d/0Bxb6U-RiSYsgQ1pTU0FsTG1SY1k/view?usp=sharing

Component cell strain & 96 well plate & Plate reader

Component cell strain

Brand: 康为世纪 (cwbiotech)

Strain: E.coli K-12 DH5α

96 well plate

Brand: Corning

-Black plate

-Flat-bottomed wells

Plate reader

Brand: BioTek

Instrument model: Cytation 5

Serial number: 1511021F

Pathlength correction: No

Number of flashes per well: 10

Orbital averaging (mm): 3.5

Fluorescence reading: top optic

Filter: Yes

Excitation wavelength (nm): 485/9

Emission wavelength (nm): 528/9

Protocol

(Some of the following steps are different from standard protocol from iGEM authority, the changes and the reasons are shown as red.)

Calibration —— OD600 reference point

Materials

1ml LUDOX

ddH₂O

96 well plate

Method

◻ Add 100 µl LUDOX into wells A1, B1, C1, D1

◻ Add 100 µl of H₂O into wells A2, B2, C2, D2

◻ Measure absorbance 600 nm of all samples in all standard measurement modes in instrument

◻ Record the data in the table below

◻ Import data into Excel (OD600 reference point tab) Sheet_1 provided

Calibration —— fluorescein fluorescence standard curve

Materials

fluorescein

10mL 1X PBS

96 well plate

Method

——Prepare the fluorescein stock solution

◻ Spin down fluorescein stock tube to make sure pellet is at the bottom of tube.

(5000rpm, 5min)

◻ Prepare 2X fluorescein stock solution (100 µM) by resuspending fluorescein in 1 mL of 1X PBS.

◻ Dilute the 2X fluorescein stock solution with 1X PBS to make a 1X fluorescein solution and resulting concentration of fluorescein stock solution 50 µM.

——Prepare the serial dilutions of fluorescein

◻ Add 100 µl of PBS into wells A2, B2, C2, D2....A12, B12, C12, D12

◻ Add 200 µl of fluorescein 1X stock solution into A1, B1, C1, D1

◻ Transfer 100 µl of fluorescein stock solution from A1 into A2.

◻ Mix A2 by pipetting up and down 3x and transfer 100 µl into A3…

◻ Mix A3 by pipetting up and down 3x and transfer 100 µl into A4...

◻ Mix A4 by pipetting up and down 3x and transfer 100 µl into A5...

◻ Mix A5 by pipetting up and down 3x and transfer 100 µl into A6...

◻ Mix A6 by pipetting up and down 3x and transfer 100 µl into A7...

◻ Mix A7 by pipetting up and down 3x and transfer 100 µl into A8...

◻ Mix A8 by pipetting up and down 3x and transfer 100 µl into A9...

◻ Mix A9 by pipetting up and down 3x and transfer 100 µl into A10...

◻ Mix A10 by pipetting up and down 3x and transfer 100 µl into A11...

◻ Mix A11 by pipetting up and down 3X and transfer 100 µl into liquid waste

◻ Repeat dilution series for rows B, C, D

◻ Measure fluorescence of all samples in all standard measurement modes in instrument

◻ Record the data in your notebook

◻ Import data into Excel (fluorescein standard curve tab) Sheet_1 provided

Cell measurement

Materials

E.coli K-12 DH5α component cells

LB (Luria Bertani) medium

Chloramphenicol (stock concentration 30 mg/mL dissolved in EtOH - working stock 30 ug/mL)

(This concentration refers to one of our PIs, and it’s the common concentration in my lab.)

(The file can be download here:

https://drive.google.com/open?id=0Bxb6U-RiSYsgbjEzU0o4eVNYNWc)

14 mL polypropylene round-bottom tube with aluminum foil cover

(Comparing with 50mL falcon tube, it is much more suitable for incubation, the reason is that its cover isn’t completely closed which can make the oxygen entry freely, so the E.coli can be incubated in an aerobic environment.)

Highlight

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Tooltips

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Dropcaps

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Blockquote

Fusce lacinia tempor malesuada. Ut lacus sapien, placerat a ornare nec, elementum sit amet felis. Maecenas pretium lorem hendrerit eros sagittis fermentum. Donec in ut odio libero, at vulputate urna. Nulla tristique mi a massa convallis cursus. Nulla eu mi magna. Etiam suscipit commodo ad gravida. Cras suscipit, quam vitae adipiscing faucibus, risus nibh laoreet odio, a porttitor metus eros ut enim. Morbi augue velit, tempus mattis sum dignissim nec, porta sed risus. Donec eget magna eu lorem tristique pellentesque eget eu dui. Fusce lacinia tempor malesuada. Ut lacus sapien, po anemat ornare nec, elementum sit amet felis. Maecenas pretium hendrerit fermentum lacus sapien, placerat a ornare nec fringilla libero convals.

Nulla nunc dui, tristique in semper vel, congue sed ligula. Nam dolor ligula, faucibus id sodales in, auctor fringilla libero. Pellentesque pellentesque eget tempor tellus. Fusce lacinia tempor malesuada. Ut lacus sapien, placerat a ornare nec, elementum sit amet felis. Maecenas pretium lorem hendrerit eros sagittis fermentum.

Fusce lacinia tempor malesuada. Ut lacus sapien, placerat a ornare nec, elementum sit amet felis. Maecenas pretium lorem hendrerit eros sagittis fermentum. Donec in ut odio libero, at vulputate urna. Nulla tristique mi a massa convallis cursus. Nulla eu mi magna. Etiam suscipit commodo ad gravida. Cras suscipit, quam vitae adipiscing faucibus, risus nibh laoreet odio, a porttitor metus eros ut enim. Morbi augue velit, tempus mattis sum dignissim nec, porta sed risus. Donec eget magna eu lorem tristique pellentesque eget eu dui. Fusce lacinia tempor malesuada. Ut lacus sapien, po anemat ornare nec, elementum sit amet felis. Maecenas pretium hendrerit fermentum lacus sapien, placerat a ornare nec fringilla libero convals.