Team:SYSU-Software/Interlab
Overview
This study aims to collect data from iGEM teams all over the world on the fluorescence of three genetic devices expressing GFP. The devices differ in their strength of the promoter. Below, we detail our Interlab Study experience and discuss what our findings could mean.
We built applicable constructs using standard biobrick assembly and we introduced the three constructs as well as positive and negative controls into Escherichia coli strain DH5α cells. After an overnight cultivation, the fluorescence and OD600 of the liquid cultures were measured using a plate reader.
Experimental Design
We built applicable constructs using standard biobrick assembly and we introduced the three constructs into Escherichia coli strain DH5α cells. The correct identity of the resulting constructs was confirmed by sequencing. Colonies were grown on appropriate selection and then screened for successful ligation and transformation by colony PCR. Positive colonies were then re-grown in LB medium in a shaking incubator. After 14 hours of cultivation, liquid cultures were diluted with LB medium in a ratio of 1:9 and then cultivated until OD600 reached a value around 0.5. 200µl of the medium were taken out and OD600 and fluorescence of the samples were measured with a plate reader.
Method & Materials
A: Cultivation
The cultivation of our bacteria was performed in 10 ml test tube with 5 ml LB medium. The cultures were kept at 37°C and 250 rpm shaking frequency. Appropriate antibiotics were added to each media (Ampicillin for I13504, chloramphenicol for J23101, J23106 and J23117, R0040 and I20270).
B: Constructs and strains
The negative control (R0040) and the positive control (I20270) were taken directly from the iGEM 2015 distribution plates. The constructs J23101+I13504, J23106+I13504 and J23117+I13504 were made using the 3A Assembly, in the original plasmid backbone of J23101, J23106 J23117. All constructs used were transformed into Escherichia coli strain DH5α cells. After overnight cultivation, the plasmids were extracted using the Omega EZNA plasmid mini kit I. The purified plasmids J23101, J23106 and J23117 were cut with the restriction enzymes SpeI and PstI, while I13504 was cut with XbaI and PstI (fig 1). Then, we run an electrophoresis to obtain the specific DNA segment using gel extraction kit. Afterwards, the specific DNA segments were ligated together using the T4 DNA Ligase.
The correct identity of the resulting constructs was firstly confirmed by a colony PCR (fig 2, 3, 4) and then was confirmed by the sequencing.
Photos
fig.1 DNA segment of restriction digest, J23101, J23106, J23117 and I13504 are shown from left to right respectively. DS5000 is used as marker.
fig.2 Colony PCR : J23101+I13504. DS5000 is used as marker.
fig.3 Colony PCR : J23106+I13504. DS5000 is used as marker.
fig. 4 Colony PCR : J23117+I13504. DS5000 is used as marker.
C: Sampling
We chose test tube to culture our bacterial which had the target plasmid and used the 96-hole flask for the plate reader’s measurement. After 14 hours of cultivation, liquid cultures were diluted with LB medium in a ratio of 1:9 and then cultivated until OD600 reached a value around 0.5. Each well of the plate contains 200µl of medium.
D: Measurement of OD using a plate reader
For OD measurement and the fluorescence measurement, Tecan Infinite © 200 PRO NanoQuant was used. Each measurement occurred in a technical triplicate with a setting of three parallel wholes and we used LB medium with antibiotics as our blank.
Devices Measured
Device 1: J23101 + I13504 (B0034-E0040-B0015), in pSB1C3
Device 2: J23106 + I13504 (B0034-E0040-B0015), in pSB1C3
Device 3: J23117 + I13504 (B0034-E0040-B0015), in pSB1C3
J23101: 2015 Kit Plate 1, Well 20K, pSB1C3
J23106: 2015 Kit Plate 1, Well 22A, pSB1C3
J23117: 2015 Kit Plate 1, Well 22K, pSB1C3
I13504: 2015 Kit Plate 4, Well 21J, pSB1A2
Appendix
Appendix 1-Sequencing Data
Appendix 2-Protocols
Results
