Difference between revisions of "Part:BBa K3036006"

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GFP with degradation conduction tag RepA

We design a device for increasing the degradation rate of green fluorescent protein (GFP) by adding a 16-amino-acid-long tag replication protein A (RepA) at the N-terminal, therefore the green fluorescence will degrade sooner when expression ends. Hence, when GFP is used as a reporter, it is more sensitive in monitoring the state of any inducible promoter.

In our project, the RepA-GFP part is used in the report plasmid to present the state of the promoter flanked by recombination sites in a more sensitive way, in order to test our bilateral switch. It enlightens us to make the same improvement with our recombination directionality factor (RDF), so as to raise efficiency of the bilateral switch.

Biology and Usage

We design a device for increasing the degradation rate of green fluorescent protein (GFP) by adding a 16-amino-acid-long tag replication protein A (RepA) at the N-terminal, therefore the green fluorescence will degrade sooner when expression ends. Hence, when GFP is used as a reporter, it is more sensitive in monitoring the state of any inducible promoter.

Green fluorescent protein (GFP) is used as a reporter to provide an easily-observed characterization. Tag RepA is used to accelerate the degradation of GFP to display the real-time data more efficiently. It is a hexadecapeptide which leads to the recognition of the protein fused with it by Clp ATPase component, therefore makes the protein degraded by Clp proteases. In this part, RepA is attached to the N-terminal of the GFP sequence to avoid the deletion of tag led by nonsense mutation.

GFP is derived from jellyfish Aequeora Victoria and we extracted its certain sequence from 2019 Distribution kit. RepA tag is derived from proteins which could be recognized by an enzyme relevant to degradation. We add this tag to GFP by elongating the primers from N-terminal.

In our project, the RepA-GFP part is used in the report plasmid to present the state of the promoter flanked by recombination sites in a more sensitive way, in order to test our bilateral switch. It enlightens us to make the same improvement with our recombination directionality factor (RDF), so as to raise efficiency of the bilateral switch.

Properties and Improvement

We construct a system where the lac promoter controls downstream GFP gene as the control group, and RepA-GFP gene as experimental group. After induced by 1 mmol/L IPTG for 4 hours the inducer is removed for next the 3 hours, their GFP fluorescence intensity and OD600 are tested over time. As is shown in Fig 1, after corrected with OD600, the average fluorescence intensities of both groups are approximately the same induced by IPTG for the first 240 minutes. However, the AFI shows a remarkable increase without induction, demonstrating that RepA-GFP indeed degrades faster.

Experimental approach

1. Transform two kinds of plasmids into E. coli DH5α competent cells respectively. System where the lac promoter controls downstream GFP gene is taken as the control group, and the RepA-GFP gene instead is taken as experimental group.
2. Divide experimental group and control group into three copies as parallel experiments (6 culture systems totally) and each copy is cultivated in 30ml LB-ampicillin medium (0.1% ampicillin) under the conditions of 37℃，200rpm in incubator overnight.
3. Dilute them to a concentration as their OD600 values equal to about 2.
4. Add 100μl 300nM IPTG (isopropyl-β-D-thiogalactopyranoside) into every culture system.
5. Samples of them (200μl each, the same below) are taken every 30min for 4 hours.
6. Remove the induction of IPTG by centrifuging the culture and wash the cells with inducer-free LB-ampicillin medium twice, note that it should be finished within 30 minutes and then take the next samples.
7. Take samples every 30min in the same manner for 3 hours.
8. Pipette 100ul culture solution of each group and mix with 100ul LB-ampicillin in 96-well plate with pure LB-ampicillin as blank to measure GFP florescence intensity and OD600 by microplate reader. The ratio of the former to the latter is used to represent their average fluorescence intensity.
9. Three repicas are tested in each group.
10. Obtain and analyze data.

Reference

[1] Butz M., Neuenschwander M., Kast P., Hilvert D.. An N-Terminal Protein Degradation Tag Enables Robust Selection of Highly Active Enzymes [J]. Biochemistry, 2011, 50(40):8594-8602.
[2] Dıaz-Lopez, T., Lages-Gonzalo, M., Serrano-Lopez, A., Alfonso, C., Rivas, G., Dıaz-Orejas, R., Giraldo, R. Structural changes in RepA, a plasmid replication initiator, upon binding to origin DNA [J]. Journal of Biological Chemistry, 2003, 278: 18606−18616.
[3] Hoskins, J. R., Wickner, S.. Two peptide sequences can function cooperatively to facilitate binding and unfolding by ClpA and degradation by ClpAP [J]. Proceedings of the National Academy of Sciences of the United States of America, 2006, 103: 909−914.

improve BBa_E0040
We imporved BBa_E0040

Sequence and Features