Part:BBa_K4054009
eGFP(enhanced green fluorescent protein) with N-terminal degradation tag RepA
In this year, we designed a composite part for adding the self-degration function to eGFP by bind a 16-amino-acid long tag called replication protein A(RepA) at the N-terminal of eGFP. The expressed eGFP will be continuely degraded by ClpAP in vivo or in vitro. Therefore, the moifed eGFP will be more specifcally reflect the state of promoter, when it works ad a repoter gene.
For us, we designed this part, RepA-eGFP, for observe the signal in real time and improve the specificity of our detecton, as they will not accumulate in our system. It also provide the availible method to improve the LuxR (an activator) we used in our project, to form a self-regulated device.
Usage and Biology
eGFP (enhanced green flourescent protein) is commonly used as a reporter in several quilified ot quantified meansurement. RepA tag could be used for increase the speed of degradation of teagged protein. It helps the recognation by ClpA enzyme and then ClpA could send the specific protein to ClpP for degradation. Meanwhile. the N-terminal tagged could avoid the structual effect around the C-terminal.
For us, we design a xomposite part for RepA-eGFP experssion in vivo, we chose the BL21(DE3) as the chassis and which contains the gene coding ClpAP, which means it could rapidly degrade the RepA-eGFP in cells. <p>Firstl, we use cell free and BL21 to express the RepA eGFP to confirm that the added tag will not affect the common function fo eGFP.
As Figure 1 shows, the expression product performs a peak of ezcitation around 488 nm and a peak of emission around 510 nm, which indicated that the RepA-eGFP was sucucessfully expressed while it remain the original charasteristics of none-tagged eGFP.
Improved by XJTLU-CHINA 2021
In order to confirm that the RepA-eGFP actually obtained the higher degradation rate, we transformed the plasmid with eGFP and RepA-eGFP into BL21(DE3) to confirm that.As the expression of the eGFP could be induced by IPTG,we controled the expression of eGFP and RepA-eGFP.in vitro . We exclude the effect of cell growth by correct fluorenscence intensity with OD600 of E,coli. We treated both types of transformed bacteria with IPTG overnight, Then, we removed the IPTG by washing several times with pure LB and start measurement the fluorenscence intensity by platereader after 3 hours culturing.
Simplified Protocol
1. Transform plasmids into E. coli BL21 (DE3) competent cells. The group with none-tagged eGFP was set as control group.
2. Plate the transformed BL21(DE3) on the LB Agar plate with Ampcillin under the conditions of 37℃ incubator overnight for selection.
3. Select the single colonies and pick them into 10mL liquid LB, and culturing under 37℃ in shaker with 180RPM overnight.
4. Add 1.74μL 300nM IPTG (isopropyl-β-D-thiogalactopyranoside) into every culture system and culture them under 18℃ in shaker with 130RPM overnight.
5. Remove the IPTG by centrifuging the bacteria solution and wash the E.coli with inducer-free LB-ampicillin medium three times as quickly as possible.
7. After 3 huors culturing in 37℃ in shaker with 180RPM，seperate the bacteria in to 96-wells plate and keep culturing in microplate reader in with 180RPM shaking.Meanwhile, detect the OD600 and emission of 510nm under 480 nm excitating light in every 2 minutes.
9. Obtain and analyze data.
Reference
Sathyanarayanan, G., Järvinen, P. and Sikanen, T.M., 2018. Quantification of digital microfluidic fluorescence assays with the Varioskan LUX Multimode Microplate Reader.
Butz, M., Neuenschwander, M., Kast, P. and Hilvert, D., 2011. An N-terminal protein degradation tag enables robust selection of highly active enzymes. Biochemistry, 50(40), pp.8594-8602.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
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