We built 3 devices (Fig. 1, Fig. 2, Fig. 3) according to Interlab study instructions. First designed in software (Geneious v. 7.0.6). Then made in our wet lab using the Biobrick Distribution Kits and confirmed by electrophoresis gel analytic digestion as well as sequencing. Finally, stocked as a glycerol stock and used for the measurements of GFP expression.
Team:Paris Bettencourt/Project/Interlab Study
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- | <div class=text><p>We built 3 devices (Fig. 1, Fig. 2, Fig. 3) according to <a href"https://2014.igem.org/Tracks/Measurement/Interlab_study">Interlab study instructions</a>. First designed in software (Geneious v. 7.0.6). Then made in our wet lab using the Biobrick Distribution Kits and confirmed by | + | <div class=text><p>We built 3 devices (Fig. 1, Fig. 2, Fig. 3) according to <a href"https://2014.igem.org/Tracks/Measurement/Interlab_study">Interlab study instructions</a>. First designed in software (Geneious v. 7.0.6). Then made in our wet lab using the Biobrick Distribution Kits and confirmed by electrophoresis gel analytic digestion as well as sequencing. Finally, stocked as a glycerol stock and used for the measurements of GFP expression. </p></div> |
<div class=image id=firstimg><img src="https://static.igem.org/mediawiki/2014/9/91/Device_1.png"><p class=definition><b>Figure 1. Device 1.</b> Geneious version 7.0.6 created by Biomatters. Available from http://www.geneious.com/</p></div> | <div class=image id=firstimg><img src="https://static.igem.org/mediawiki/2014/9/91/Device_1.png"><p class=definition><b>Figure 1. Device 1.</b> Geneious version 7.0.6 created by Biomatters. Available from http://www.geneious.com/</p></div> | ||
</br><h6>Device 1</h6></br> | </br><h6>Device 1</h6></br> |
Latest revision as of 16:05, 7 December 2014
![](https://static.igem.org/mediawiki/2014/8/8d/PB14interlab_study.png)
Devices | Sequencing | Conclusion |
Achievements
Photo 1. Fluorescent devices and LB control. A photo of the three devices and an LB control was taken with a help of transillumiantor
- 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
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 transformation and ligation techniques for the inexperienced synthetic biologist on the team.
Devices
![](https://static.igem.org/mediawiki/2014/9/91/Device_1.png)
Figure 1. Device 1. Geneious version 7.0.6 created by Biomatters. Available from http://www.geneious.com/
Device 1
![](https://static.igem.org/mediawiki/2014/9/91/Device_1.png)
Figure 1. 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
We followed iGEM Distribution Kit instructions to extract DNA from the Biobrick Plate 2, Well 17F (2012) and then Heat Shock transformed the E. coli colonies that grew in the selective Kanymycin were grown in liquid media and made into glycerol stocks labelled G.22.
![](https://static.igem.org/mediawiki/2014/0/02/Device_2.png)
Figure 2. Device 2.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
We followed the iGEM Distribution Kit instructions to extract DNA from the Biobrick BBa_K823005 and BBa_E0240 and then Heat Shock transformed E. coliThe colonies that survived after selection in choloamphenicol were cultured overnight. We used 750uL of the liquid cultures for a glycerol stock . The remaining 4.25 mL were used to make minipreps . We measured DNA content with the nanodrop. Digestion analysis:
- 5 ug plasmid
- 5 ul Buffer
- 2.5 uL SpeI + 2.5 uL PstI (BBa_K823005) / 2.5 uL XbeI + 2.5 uL PstI (BBa_E0240)
- Complete with H2O
- 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
![](https://static.igem.org/mediawiki/2014/d/d9/Device_3.png)
Figure 3. Device 3.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 mismatched basepairs.
BBa_J23115 + BBa_E0240 (B0032-E0040-B0010-B0012), in the pSB1C3 vector. Selection marker : Chloramphenicol Promoter expected sequence : tttatagctagctcagtcctaggtacaatgctagc (mismatched 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 proceeded 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.
1. Device 1
Promoter expected sequence : tttacagctagctcagtcctaggtattatgctagc Sequenced device’s promoter: TTTACAGCTAGCTCAGTCCTAGGTTATTATGCTAGC Complete sequenced device: ATTTGTATCTTACTATAAATAGGCGTATCACGAGGCACGAAATTTCAGATAAAAAAAATCCTTAGCTTTCGCTAAGGATGATTTCTGGAATTCGCGAGCCGCTTCTAGAGATTTACAGCTAGCTCAGTCCTAGGTTATTATGCTAGCTACTAGAGTTCACACAGGAAAGTACTAGATGCGTAAAGGAGAAGAACTTTTCACTGGAGTTGTCCCAATTCTTGTTGAATTAGATGGTGATGTTAATGGGCACAAATTTTCTGTCAGTGGAGAGGGTGAAGGTGATGCAACATACGGAAAACTTACCCTTAAATTTATTTGCACTACTGGAAAACTACCTGTTCCATGGCCAACACTTGTCACTACTTTCGGTTATGGTGTTCAATGCTTTGCGAGATACCCAGATCATATGAAACAGCATGACTTTTTCAAGAGTGCCATGCCCGAAGGTTATGTACAGGAAAGAACTATATTTTTCAAAGATGACGGGAACTACAAGACACGTGCTGAAGTCAA...
2. Device 2
Promoter expected sequence : tttacagctagctcagtcctaggtattatgctagc Sequenced device’s promoter: TTTACAGCTAGCTCAGTCCTAGGTATTATGCTAGC Complete sequenced device: TTTGATAACTATAAATAGGCGTATCACGAGGCAGAATTTCAGATAAAAAAAATCCTTAGCTTTCGCTAAGGATGATTTCTGGAATTCGCGGCCGCTTCTAGAGTTTACAGCTAGCTCAGTCCTAGGTATTATGCTAGCTACTAGAGTCACACAGGAAAGTACTAGATGCGTAAAGGAGAAGAACTTTTCACTGGAGTTGTCCCAATTCTTGTTGAATTAGATGGTGATGTTAATGGGCACAAATTTTCTGTCAGTGGAGAGGGTGAAGGTGATGCAACATACGGAAAACTTACCCTTAAATTTATTTGCACTACTGGAAAACTACCTGTTCCATGGCCAACACTTGTCACTACTTTCGGTTATGGTGTTCAATGCTTTGCGAGATACCCAGATCATATGAAACAGCATGACTTTTTCAAGAGTGCCATGCCCGAAGGTTATGTACAGGAAAGAACTATATTTTTCAAAGATGACGGGAACTACAAGACACGTGCTGAAGTCAAGTTTGAAGGTGATACCCTTGTTAATAGAATCGAGTTAAAAGGTATTGATTTTAAAGAAGATGGAAACATTCTTGGACACAAATTGGAATACAACTATAACTCACACAATGTATACATCATGGCAGACAAACAAAAGAATGGAATCAAAGTTAACTTCAAAATTAGACACAACATTGA...
3. Device 3
Promoter expected sequence : tttatagctagctcagtcctaggtacaatgctagc Sequenced promoter sequence:TTTATAGCTAGCTCAGTCCTAGGTACAATGCTAGC Complete sequence: GACTCTGATAACTATAAATAGGCGTATCACGAGGCAGAATTTCAGATAAAAAAAATCCTTAGCTTTCGCTAAGGATGATTTCTGGAATTCGCGGCCGCTTCTAGAGTTTATAGCTAGCTCAGTCCTAGGTACAATGCTAGCTACTAGAGTCACACAGGAAAGTACTAGATGCGTAAAGGAGAAGAACTTTTCACTGGAGTTGTCCCAATTCTTGTTGAATTAGATGGTGATGTTAATGGGCACAAATTTTCTGTCAGTGGAGAGGGTGAAGGTGATGCAACATACGGAAAACTTACCCTTAAATTTATTTGCACTACTGGAAAACTACCTGTTCCATGGCCAACACTTGTCACTACTTTCGGTTATGGTGTTCAATGCTTTGCGAGATACCCAGATCATATGAAACAGCATGACTTTTTCAAGAGTGCCATGCCCGAAGGTTATGTACAGGAAAGAACTATATTTTTCAAAGATGACGGGAACTACAAGACACGTGCTGAAGTCAAGTTTGAAGGTGATACCCTTGTTAATAGAATCGAGTTAAAAGGTATTGATTTTAAAGAAGATGGAAACATTCTTGGACACAAATTGGAATACAACTATAACTCACACAATGTATACATCATGGCAGACAAACAAAAGAATGGAATCAAA...
![](https://static.igem.org/mediawiki/2014/0/0a/OD600_min.jpg)
![](https://static.igem.org/mediawiki/2014/e/e7/Fluo_samples_min.jpg)
![](https://static.igem.org/mediawiki/2014/d/d0/Fluo_od_min.jpg)
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 Devices (Fig. 4). 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) as can be seen in the Fig. 5 & Fig. 6