Team:Amoy/Interlab

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Project

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INTERLAB

1. 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.This year,three devices were cloned by ourselves, and one positive control and one negative control were provided by the registry.Using a plate reader, fluorescence measurements were obtained in arbitrary units. The results show increased fluorescence in the stronger promoter expected.

After the experiment, three required devices were created:
J23101+I13504 in the pSB3K3 backbone.
J23106+I13504 in the pSB1C3 backbone.
J23117+I13504 in the pSB1C3 backbone.

Devices constructed in pSB1C3 are high copy number plasmids, hence a strong fluorescence can be obviously observed by naked eyes. While for device J23117 + I13504, the promoter strength is the weakest, we could barely observe any fluorescence under natural light.

Figure1.GFP generator with different promoters under natural light.1:J23101+I13504 in DH5α; 2：J23106+i13504 in DH5α;3:J23117+I13504 in DH5α.

Figure2.Centrifugation of bacteria. 1:J23101+I13504; 2:J23106+I13504; 3:J23117+I13504; 4.positive control(I20270); 5.negative control(R0040 in PSB1C3)

Digestion Verification

Four devices(J23101+I13504/J23106+I13504/J23117+I13504/I20270) are double digested at EcoRI and PstI restriction sites to verify the target parts. The restriction map is shown as Figure3. As expected,the second bands are supposed to be aroud 1000bp,which is consistent with what we saw on the map.

Figure3. Restriction map of digestion verification. 2000plus DNA marker.

1.Double digestion of J23101+I13504(constructed plasmid)
2.Double digestion of J23106+I13504(constructed plasmid)
3.Double digestion of J23117+I13504(constructed plasmid)
4.Double digestion of I20270 in PSB1C3 backbone(provided in registry)

DNA sequencing

All created parts are verified by DNA sequencing,as shown in figure4.

Figure4. DNA sequencing results of three required devices, analyzed by DNAMAN software.

2. Protocol

1. Transform constructed plasmids into DH5α competent cells, grown in incubator for 12 hrs at 37℃.
2. Add 5 mL LB medium with antibiotic (Chloramphenicol 35μg/ml) into test tubes, choose monoclonal cells from the petri dish.
3. Set up 3 biological replicates of each device.Cultures were grown in test tubes for 16 hours at 37℃,shaking at 200 rpm.