Difference between revisions of "Part:BBa K4054009"

Latest revision as of 01:05, 22 October 2021

eGFP(enhanced green fluorescent protein) with N-terminal degradation tag RepA

In this year, we designed a composite part for adding the self-degradation function to eGFP (BBa_S03452)by bind a 16-amino-acid long tag called replication protein A(RepA) at the N-terminal of eGFP. The expressed eGFP will be continuously degraded by ClpAP in vivo or in vitro. Therefore, the modified eGFP will more specifcally reflect the state of the promoter, when it works as a reporter gene.

For us, we designed this part, RepA-eGFP, for visualizing the signal and improving the specificity of our detection, as it will not accumulate in our system and give a false positive signal after long-term expression induced by low level of inducer. It also provides an available method to improve the LuxR (an activator) we used in our project to form a synthesis/degradation bistable system.

Usage and Biology

eGFP (enhanced green fluorescent protein) is commonly used as a reporter protein in numerous qualitative or quantitative measurements. RepA tag could be used to increase the rate of degradation of the tagged proteins. It helps the recognition by the ClpA subunit of the enzyme which denatures and sends the tagged protein to ClpP subunit for degradation. Meanwhile. the N-terminal tagged could avoid the structural effect around the C-terminal.

For us, we designed a composite part for RepA-eGFP expression 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.

Firstly, we use cell-free system and BL21 to express the RepA-eGFP to confirm that the added tag will not affect the common function of eGFP.

Figrure 1.The spectrum of emission and excitation of expressed RepA-Egfp.

As Figure 1 shows, the expression product displays a peak of excitation around 488 nm and a peak of emission around 510 nm, which indicates that the RepA-eGFP was successfully expressed while it remained the original characteristics of none-tagged eGFP.

Improved by XJTLU-CHINA 2021

We transformed the plasmid with eGFP and RepA-eGFP into BL21(DE3) to confirm that the RepA-eGFP obtained the higher degradation rate.As the expression of the eGFP could be induced by IPTG,we could regulate the expression of eGFP and RepA-eGFP.in vitro . We excluded the effect of cell growth by normalizing fluorescence 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 started measurement the fluorescence intensity by plate reader after 3 hours culturing.

Figure 2. The trend of expression rate with the changes of LuxR concentration.

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 Ampicillin under the conditions of 37℃ in incubator overnight for selection.
3. Select the single colonies and pick them into 10mL liquid LB, and culture 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.
6. After 3 hours culturing in 37℃ in shaker with 180RPM，separate the bacteria into 96-wells plate and keep culturing in microplate reader in with 180RPM shaking. Meanwhile, measure the OD600 and emission of 510nm under 480 nm exciting light in every 2 minutes.
7. 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