Team:Paris Bettencourt/Project/Interlab Study

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@iGEM_Paris

BACKGROUND

The first international interlab measurement study in synthetic biology!

AIMS

Measure GFP expression under different Anderson promoters and in different plasmids.

RESULTS

- Successfully built the three devices
- Sequenced and gel verified the three devices
-Characterised all the three devices with the OD600 and fluorescence measurements in a Tecan micro-plate reader
- Reported the results on the Interlab Study page of the iGEM wiki
- Completed the description of the BBa_I20260 - Device 1
- Submitted and send parts: BBa_K1403000 - Device 2 and BBa_K1403001 - Device 3 to the Biobricks registry

Devices

Sequencing

Conclusion

Introduction

"The goal of the interlab study is to obtain fluorescence data for three specific genetic devices expressing GFP from iGEM teams around the world. "

Motivation

iGEM Paris Bettencourt team supported this initiative with pleasure. We strongly believe that this study will help to define standards for BioBrick constructs and measure the difference between different labs and measurement methods. It was also a great training for inexperienced synthetic biologist of the team which allowed as to train transformation and ligation techniques.

Devices

We build 3 devices according to Interlab study instructions. First designed in a software (Geneious v. 7.0.6). Then realised in our wet lab out of Biobrick Distribution Kits. Confirmed by eletrophoresis gel analytic dgestion as well as sequencing. Finally, stocked as a glycerol stock and used for the measurments of GFP expression.

Geneious version 7.0.6 created by Biomatters. Available from http://www.geneious.com/

Device 1

Geneious version 7.0.6 created by Biomatters. Available from http://www.geneious.com/

BBa_I20260 (J23101-B0032-E0040-B0010-B0012) in the pSB3K3 vector.

Selection marker : Kanamycin

Promoter expected sequence : tttacagctagctcagtcctaggtattatgctagc

2012 BioBrick Kit location

BBa_I20260: Plate 2, Well 17F

I followed iGEM Distribution Kit instructions to extract DNA from the Biobrick Plate 2, Well 17F (2012) and then Heat Shock transformation of E.coli
For successful Kanymycin plates I prepared glycerol stock and labbeled it G.22.

Geneious version 7.0.6 created by Biomatters. Available from http://www.geneious.com/

Device 2

Our part in the registry: BBa_K1403000

BBa_J23101 + BBa_E0240 (B0032-E0040-B0010-B0012), in the pSB1C3 vector.
Selection marker : Chloramphenicol
Promoter expected sequence : tttacagctagctcagtcctaggtattatgctagc

2014 Biobrick Kit locations

BBa_K823005 (BBa_J23101 in pSB1C3): Plate 1, Well 20K
BBa_E0240 (in pSB1C3): Plate 2, Well 24B

I followed iGEM Distribution Kit instructions to extract DNA from the Biobrick BBa_K823005 and BBa_E0240 and then Heat Shock transformation of E.coli
For successful Chloramphenicol plates, form single colonies I prepared liquid cultures overnight. I used 750uL of the liquid cultures for a glycerol stock . I used remaining 4,25 mL to make minipreps . I measured DNA content with the nanodrop.

Digestion analysis:

- 5 ug plasmid
- 5 ul FD Buffer
- 2.5 uL SpeI + 2.5 uL PstI (BBa_K823005) / 2.5 uL XbeI + 2.5 uL PstI (BBa_E0240)
- complete with H2O

(Final volume of 50 uL)
We made an eletrophoresis gel to check the fragments (the bands at around 876 bp for GFP and 2100 bp for the promoter + backbone)and then extract BBa_E0240 with Gel extraction kit. For the plasmid with the promoter I used a PCR purification kit. I introduced the GFP fragment to the Plasmid + backbone through ligation of the sticky ends SpeI and XbeI. Quantified DNA in two parts with nanodrop. The amount of vector:insert has been calculated with Promega calculator.

5X Ligase Reaction Buffer 4 μl
Insert: Vector Molar Ratio 1:1, 1:3, 1:5
Total DNA 0.01-0.1 μg
T4 DNA Ligase 1 uL
Autoclaved distilled water to 25uL
Incubate at 22°C for 1h
16°C overnight

I transformed the ligation product following Heat Shock transformation of E.coli. I have put a single colony into a liquid culture with the appropriate antibiotic and the next day I prepared a glycerol stock .

Geneious version 7.0.6 created by Biomatters. Available from http://www.geneious.com/

Device 3

Our part in the registry: BBa_K1403001

*BBa_J23115 was cloned using BBa_K823012 and therefore should have 2 missmatched basepairs.

BBa_J23115 + BBa_E0240 (B0032-E0040-B0010-B0012), in the pSB1C3 vector.
Selection marker : Chloramphenicol
Promoter expected sequence : tttatagctagctcagtcctaggtacaatgctagc (missmatched basepairs compared to real BBa_J23115 are underlined)

2014 Biobrick Kit locations

BBa_K823012 (BBa_J23115 in pSB1C3): Plate 1, Well 22I
BBa_E0240 (in pSB1C3): Plate 2, Well 24B

In order to prepare the third device we proceed exactly in the same way as for the Device 2, except we used BBa_K823012 instead of BBa_K823005

Sequencing

We sequenced all the three devices using iGEM Verification Primers (forward). Sequences are matching expected constructs.

First Device

...GAAACAGCATGACTTTTTCAAGAGTGCCATGCCCGAAGGTTATGTACAGGAAAGAACTATATTTTTCAAAGATGACGGGAACTACAAGACACGTGCTGAAGTCAAGTTTGAAGGTGATACCCTTGTTAATAGAATCGAGTTAAAAGGTATTGATTTTAAAGAAGATGGAAACATTCTTGGACACAAATTGGAATACAACTATAACTCACACAATGTATACATCATGGCAGACAAACAAAAGAATGGAATCAAAGTTAACTTCAAAATTAGACACAACATTGAAGATGGAAGCGTTCAACTAGCAGACCATTATCAACAAAATACTCCAATTGGCGATGGCCCTGTCCTTTTACCAGACAACCATTACCTGTCCACACAATCTGCCCTTTCGAAAGATCCCAACGAAAAGAGAGACCACATGGTCCTTCTTGAGTTTGTAACAGCTGCTGGGATTACACATGGCATGGATGAACTATACAAATAATAATACTAGAGCCAGGCATCAAATAAAACGAAAGGCTCAGTCGAAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCTACTAGAGTCACACTGGGCTCACCTTCGGGTGGGCCTTTCTGCGTTTATATACTAATAACCGGCCGCTGCAGTCCCGGCAAAAAAAGGGCAAAGGTGTCCACCA

Promoter expected sequence : tttacagctagctcagtcctaggtattatgctagc
Sequenced device’s promoter: TTTACAGCTAGCTCAGTCCTAGGTTATTATGCTAGC
Complete sequenced device:
ATTTGTATCTTACTATAAATAGGCGTATCACGAGGCACGAAATTTCAGATAAAAAAAATCCTTAGCTTTCGCTAAGGATGATTTCTGGAATTCGCGAGCCGCTTCTAGAGATTTACAGCTAGCTCAGTCCTAGGTTATTATGCTAGCTACTAGAGTTCACACAGGAAAGTACTAGATGCGTAAAGGAGAAGAACTTTTCACTGGAGTTGTCCCAATTCTTGTTGAATTAGATGGTGATGTTAATGGGCACAAATTTTCTGTCAGTGGAGAGGGTGAAGGTGATGCAACATACGGAAAACTTACCCTTAAATTTATTTGCACTACTGGAAAACTACCTGTTCCATGGCCAACACTTGTCACTACTTTCGGTTATGGTGTTCAATGCTTTGCGAGATACCCAGATCATAT...

Second Device

...AGATGGAAGCGTTCAACTAGCAGACCATTATCAACAAAATACTCCAATTGGCGATGGCCCTGTCCTTTTACCAGACAACCATTACCTGTCCACACAATCTGCCCTTTCGAAAGATCCCAACGAAAAGAGAGACCACATGGTCCTTCTTGAGTTTGTAACAGCTGCTGGGATTACACATGGCATGGATGAACTATACAAATAATAATACTAGAGCCAGGCATCAAATAAAACGAAAGGCTCAGTCGAAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCTACTAGAGTCACACTGGCTCACCTTCGGGTGGGCCTTTCTGCGTTTATATACTAGTAGCGGCCGCTGCAGTCCGGCAAAAAAGGGCAAGGTGTCACCACCCTGCCCTTTTTCTTTAAAACCGAAAAGATTACTTCCCGTTATGCAGGCTTCCTCGCTCACTGAATCGCTGCGCTCGGTCGTTCGGCTGCGGCGAACGGTATCAGCTCACTCAAAGGCGGTAATACCGGTTATCCCAAGAAATCAGGGGATAACCCCAGGAAAAAAACTTGGGACCAAAAGGCCACCCAAAGGGCCAGGAACCGTAAAAAAGGCCCCGTTTTCTGGGGTTTTTTCCAAAAGGCTCCGGCCCCCCTGGAAAAGACTTCAAAAATTCCGCCTTCTAATCCTAAAGGTGGGAAAACCCCCAA

Promoter expected sequence : tttacagctagctcagtcctaggtattatgctagc
Sequenced device’s promoter: TTTACAGCTAGCTCAGTCCTAGGTATTATGCTAGC
Complete sequenced device:
TTTGATAACTATAAATAGGCGTATCACGAGGCAGAATTTCAGATAAAAAAAATCCTTAGCTTTCGCTAAGGATGATTTCTGGAATTCGCGGCCGCTTCTAGAGTTTACAGCTAGCTCAGTCCTAGGTATTATGCTAGCTACTAGAGTCACACAGGAAAGTACTAGATGCGTAAAGGAGAAGAACTTTTCACTGGAGTTGTCCCAATTCTTGTTGAATTAGATGGTGATGTTAATGGGCACAAATTTTCTGTCAGTGGAGAGGGTGAAGGTGATGCAACATACGGAAAACTTACCCTTAAATTTATTTGCACTACTGGAAAACTACCTGTTCCATGGCCAACACTTGTCACTACTTTCGGTTATGGTGTTCAATGCTTTGCGAGATACCCAGATCATATGAAACAGCATGACTTTTTCAAGAGTGCCATGCCCGAAGGTTATGTACAGGAAAGAACTATATTTTTCAAAGATGACGGGAACTACAAGACACGTGCTGAAGTCAAGTTTGAAGGTGATACCCTTGTTAATAGAATCGAGTTAAAAGGTATTGATTTTAAAGAAGATGGAAACATTCTTGGACACAAATTGGAATACAACTATAACTCACACAATGTATACATCATGGCAGACAAACAAAAGAATGGAATCAAAGTTAACTTCAAAATTAGACACAACATTGA...

Third Device

...AAGGTATTGATTTTAAAGAAGATGGAAACATTCTTGGACACAAATTGGAATACAACTATAACTCACACAATGTATACATCATGGCAGACAAACAAAAGAATGGAATCAAAGTTAACTTCAAAATTAGACACAACATTGAAGATGGAAGCGTTCAACTAGCAGACCATTATCAACAAAATACTCCAATTGGCGATGGCCCTGTCCTTTTACCAGACAACCATTACCTGTCCACACAATCTGCCCTTTCGAAAGATCCCAACGAAAAGAGAGACCACATGGTCCTTCTTGAGTTTGTAACAGCTGCTGGGATTACACATGGCATGGATGAACTATACAAATAATAATACTAGAGCCAGGCATCAAATAAAACGAAAGGCTCAGTCGAAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCTACTAGAGTCACACTGGCTCACCTTCGGGTGGGCCTTTCTGCGTTTATATACTAGTAGCGGCCGCTGCAGTCCGGCAAAAAAGGGCAAGGTGTCACCACCCTGCCCTTTTTCTTTAAAACCGAAAAGATTACTTCGCGTTATGCAGGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCCGGGGGATAACCGCAGGAAAAAACATGTGGAGCCAAAAGGCCAACCAAAAGGCCAGGAACCGTAAAAAAGGCCCCGTTTGCTGGGGTTTTTTCCCAAGGGTCCCGCCCCCCTGGAAAAAGTTCCAA

Promoter expected sequence : tttatagctagctcagtcctaggtacaatgctagc
Sequenced promoter sequence:TTTATAGCTAGCTCAGTCCTAGGTACAATGCTAGC
Complete sequence:
GACTCTGATAACTATAAATAGGCGTATCACGAGGCAGAATTTCAGATAAAAAAAATCCTTAGCTTTCGCTAAGGATGATTTCTGGAATTCGCGGCCGCTTCTAGAGTTTATAGCTAGCTCAGTCCTAGGTACAATGCTAGCTACTAGAGTCACACAGGAAAGTACTAGATGCGTAAAGGAGAAGAACTTTTCACTGGAGTTGTCCCAATTCTTGTTGAATTAGATGGTGATGTTAATGGGCACAAATTTTCTGTCAGTGGAGAGGGTGAAGGTGATGCAACATACGGAAAACTTACCCTTAAATTTATTTGCACTACTGGAAAACTACCTGTTCCATGGCCAACACTTGTCACTACTTTCGGTTATGGTGTTCAATGCTTTGCGAGATACCCAGATCATATGAAACAGCATGACTTTTTCAAGAGTGCCATGCCCGAAGGTTATGTACAGGAAAGAACTATATTTTTCAAAGATGACGGGAACTACAAGACACGTGCTGAAGTCAAGTTTGAAGGTGATACCCTTGTTAATAGAATCGAGTTAA...

OD600 and fluorescence measure over 20h

Samples preparation: Single colonies were inoculated in 5mL LB broth with appropriate antibiotic and grown to saturation overnight (16h) at 37°C with shaking (220 rpm). Samples were diluted 100x (50um in 5 mL LB with appropriate antibiotic) and incubated for 2h at 37°C prior to measurement.

Control:
LB broth with antibiotics (chloramphenicol/kanamycin)- no fluorescence
NEB turbo without fluorescence - no fluorescence, no cells

Measurment
Greiner 96 plates were loaded with 150um of cells in LB and 30um mineral oil Cells have been diluted prior to measurement as described above. Background absorbance and fluorescence was determined from LB control.
Data from the top row were excluded due to the likely evaporation and artefacts (edge effects).

Results

Here we present the result of the measurments. The measument has been realised in a comparable growth conditions for all Divices (Fig.1). We can clearly see that the highest GFP expression levels occures under BBa_J23101 Anderson's strong promoter in a high copy plasmid sPB1C3 (Device 2: BBa_K1403000). The lowest GFP levels occures under very weak Anderson's promoter - mutated J23115 (Device 3: BBa_K1403001)

Fig.1 Mean OD600 absorbance measured over 20h. Background absorbance (LB) subtracted from the samples. Values reported in the log scale with error bars for standard deviation (7 replicates for each sample).

Fig.2 Mean of green fluorescence for three devices and NEB turbo cells.
Background fluorescence (LB) subtracted from the samples. Values reported in the log scale with error bars for standard deviation (7 replicates for Devices 1, 2, 3 and 6 replicates for NEB).

Fig.3 Mean of green fluorescence divided by optical density 600 for three devices and NEB turbo cells.
Background fluorescence (LB) subtracted from the samples. Values reported in the log scale with error bars for standard deviation (7 replicates for Devices 1, 2, 3 and 6 replicates for NEB).

Centre for Research and Interdisciplinarity (CRI)
Faculty of Medicine Cochin Port-Royal, South wing, 2nd floor
Paris Descartes University
24, rue du Faubourg Saint Jacques
75014 Paris, France

+33 1 44 41 25 22/25

paris-bettencourt-igem@googlegroups.com

@@ Line 269: / Line 269: @@
 		<div class=image id=firstimg><img src="https://static.igem.org/mediawiki/2014/9/91/Device_1.png"><p class=definition>Geneious version 7.0.6 created by Biomatters. Available from http://www.geneious.com/</p></div>
 		</br><h6>Device 1</h6></br>
+		<div class=image id=firstimg><img src="https://static.igem.org/mediawiki/2014/9/91/Device_1.png"><p class=definition>Geneious version 7.0.6 created by Biomatters. Available from http://www.geneious.com/</p></div>
 		<div class=text><p><u>BBa_I20260</u> (J23101-B0032-E0040-B0010-B0012) in the <u>pSB3K3</u> vector.</p>
 			<p>Selection marker : Kanamycin</p>