Team:TecCEM HS/Interlab

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Interlab study

Overview

As this is the first time we participate as a High School team, it was also the first time we participated in the second edition of the InterLab Study, where we were requested to measure the relative fluorescence of three devices containing a GFP as an expression marker and three different promoters of the same family.

In this section of our wiki, we will present our results, as well as the protocols we used and how we determined the device was actually assembled.

The devices were constructed with promoters from a family of constitutive promoters described by Chris Anderson (iGEM Berkeley Team, 2006). There are 20 promoters that have different efficiencies. This time we reported the relative efficiency of the following promoters:

1) Promoter BBa_K823005 in pSB1C3
2) Promoter BBa_K823008 in pSB1C3
3) Promoter BBa_K823013 in pSB1C3

We then assembled each one of them with a GFP cassette (BBa_I13504 in pSB1A2). The efficiency of the promoters is measured using a GFP because this is one of the most used expression markers, making it easy to compare relative fluorescence and test which promoters is being more efficient.

We measured the fluorescence in RFU (Relative Fluorescence Unit) with the Microplate Fluorescence and Luminiscence Reader Fluoroscan Ascent FL from Labsystems.

Protocols

Heatshock transformation of BB

1. Add 1 μl of DNA from the corresponding Kit Plate to 50 μl competent cells.
2. Incubate 30 min on ice
3. Heat shock 42ºC, 30s
4. Place samples on ice for 5 minutes
5. Add 950 μl SOC medium
6. Incubate at 37ºC for 1 hour, 250 rpm
7. Plate 200 μl of transformed cells into warm, LB agar with the proper antibiotic (kanamycin 15 mg/ml, chloramphenicol 35 mg/ml or ampicillin 100 mg/ml)
8. Incubate overnight at 37ºC (up to 16 h)
9. Isolate a single colony and culture in liquid LB broth for future extractions and plasmid isolation

Plasmid extraction by Miniprep

1. Culture 2 ml of transformed E.coli into a 50 ml flask containing LB with the proper antibiotic (0.1% v/v).
2. Incubate ON, 37ºC, 250 rpm
3. Place the flask on ice for 20 min
4. Centrifuge the 50 ml of culture at 13000 rpm for 2 minutes
5. Discard supernatant
6. Resuspend biomass in 350 μl of STET buffer
7. Transferr to a 2 ml microtube
8. Add 5 μl of lysozyme (10 mg/ml)
9. Incubate at room temperature for 5 minutes
10. Place the microtube in a boiling water bath during 2 minutes to inactivate the lysozyme.
11. Place sample immediately on ice, centrifuge at 4ºC, 13000 rpm, 10 minutes
12. Take out the bacterial pellet and add 40 μl of RNase A (200ug/ml) to the liquid phase left on the microtube
13. Incubate at room temperature for 10 minutes
14. Add 75 μl of sodium acetate (3M, pH 5.2) and 400 μl of isopropanol
15. Stirr gently and incubate for 10 minutes at room temperature
16. Centrifugeat 13500 for 10 minutes and discard the supernatant
17. Wash pellet with 1 ml of ethanol (70%) two times
18. Let pDNA drying for 45 minutes
19. Resuspend in 200 μl of nuclease-free water
20. Quantify by spectrophotometry

Device Assembly

1. BBa_I13504 digestion: Add in a 0.2 ml tube (for a 50 μl reaction):

Up to 50 μl nuclease-free water
5 μl NEB® Buffer 2.1 10X
500 μg Plasmid with BBa_I13504
1 μl PstI
1 μl XbaI

2. Promoter digestion: Add in a 0.2 ml tube (for a 50 μl reaction):

Up to 50 μl nuclease-free water
5 μl NEB® Buffer 2.1 10X
500 μg Plasmid with Promoter
1 μl PstI
1 μl SpeI

3. Mix gently each tube
4. Place at thermoblock, 37ºC for 1 hour
5. Inactivate enzymes at 80ºC for 20 minutes
6. Store digestion products at -20ºC
7. Ligation: For a 20 μl reaction. Add in the following order:

11 μl nuclease-free water
4 μl Promoter digestion
2 μl BBa_I13504 digestion
2 μl 10X T4 DNA Ligase Buffer
1 μl T4 DNA Ligase

8. Incubate at room temperature (20ºC - 25ºC) for 2 hours
9. Heat inactivate at 80ºC for 20 minutes
10. Transform 10 μl ligation into 50 μl of competent cells. Use the heatshock transformation protocol mentioned above
11. Store the rest at -20ºC

Device Verification

'''Restriction Map of Devices'''
1. Add in a 0.2 ml tube (for a 20 μl reaction):

Up to 20 μl nuclease-free water

5 μl CutSmart® Buffer 10X

500 μg Plasmid with Device

1 μl XhoI

2. Mix gently each tube
3. Place at thermoblock, 37ºC for 1 hour
4. Inactivate enzymes at 80ºC for 20 minutes
5. Run electrophoresis gel to observe restriction map results

Growing cells for Measurement

1. Streak out an agar LB plate (Cam 35 mg/ml) with the devices and strak out control. Incubate for 16 h at 37ºC.
2. Inoculate liquid culture with experimental devices and controls in conical tubes with 3 ml LB broth/Cam+. Incubate for 16 h at 37ºC, 250 rpm. * From this step everything is in triplicate.
3. Take 500ul and inoculate liquid culture with experimental devices and controls in conical tubes with 5 ml LB broth/Cam+. Incubate for 3 h at 37ºC, 250 rpm.

Plate Reader Measurement

1. Add 5 ml of PBS 1X to the conical tubes with 5ml fresh culture.
2. Centrifugate at 6000 rpm for 2 minutes.
3. Discard supernatant and add 1 ml PBS 1X, transfer to a microtube.
4. Centrifugate at 10000 rpm 2 minutes. Discard supernatant. Add 1ml PBS 1X and resuspend the pellet.
5. Obtain OD 600 of the culture with a dilution 1:10 with PBS 1X.
6. If OD is within 5% of 0.5, proceed. If not, recalculate dilution.
6. Set the plate reader to measure GFP and measure each replicate three times.

Results

Biobricks Measured

Device 1	BBa_K823005 + BBa_I13504 (B0034-E0040-B0015) + in pSB1C3
Device 2	BBa_K823008 + BBa_I13504 (B0034-E0040-B0015) + in pSB1C3
Device 3	BBa_K823013 + BBa_I13504 (B0034-E0040-B0015) + in pSB1C3
Positive Control	BBa_I20270 in pSB1C3
Negative Control	BBa_R0040 in pSB1C3