Improved by XJTLU-CHINA 2021

eGFP (enhanced green fluorescent protein) is commonly used as a reporter in numerous qualified or quantified measurements. RepA tag could be used for increase the speed of degradation of the tagged proteins. It helps the recognition 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 designed a composite part for RepA-eGFP(BBa_K4054009) 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 performs 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.

Experimental Measurement

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.

Contribution Made by Shanghai_HS 2020

BBa_K3585003 is a composite part constituting of enhanced green fluorescent protein gene under control of phoA promoter (basic parts BBa_K3527000). The similar design was seen for composite parts BBa_K1139201 and BBa_K737024. The part BBa_K737024 does not have experimental data showing its function and the part BBa_K1139201 has sufficient data showing its function but no practical application.

Figure 1

Usage and Biology

enhanced for mammalian cells. excitation wavelength 488-510 nm.

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
COMPATIBLE WITH RFC[12]
21
COMPATIBLE WITH RFC[21]
23
COMPATIBLE WITH RFC[23]
25
COMPATIBLE WITH RFC[25]
1000
COMPATIBLE WITH RFC[1000]

Measurements by KORHS team

Aim: For our characterization experiment, our team has chosen to characterize the part BBA_S03452 (Enhanced green fluorescence protein). EGFP is a type of fluorescent protein whose fluorescence measurement can be used to quantify protein. Our team focused on altering the aeration condition for EGFP (BBA_S03452) expression in three different conditions as described in the method section to investigate the effect of aeration on E.coli growth (cell line BL21(DE3)) and protein expression.

Method : We tested three experimental groups for EGFP expression. Group # 1. Regular Flask with 100 RPM Group # 2. Regular Flask with 150 RPM Group # 3. Baffled Flask with 150 RPM

Protocol : Transform the eGFP gene contained plasmid to E.coli (BL21(DE3)) and plated on LB plates with antibiotics(carbenicillin). Grow overnight at 37℃. Starter culture preparation: Prepare 100ml of LB with antibiotics and inoculate a single colony. Grow overnight at 37℃ with shaking. Prepare 2 regular flasks and 1 baffled flask with 500ml of LB. Seed 30ml of starter culture at each flask. Grow at 37℃ with using shaking incubator with different rpm. Keep measuring OD600. When OD600 reaches 0.4~0.6, add 500㎕ of 1M isopropyl-β-thiogalactopyranoside(IPTG) to the cultures to induce expression. Measure OD600 and fluorescence intensity of E.coli cells every 20 minutes.

Results :

Fluorescence Measurement of fluorescein standard and E.coli expressing EGFP

Making calibration curve using fluorescein standard from iGEM

Fluorescein provided by iGEM was used a standard for fluorescence measurement. The near linear response of fluorescence signal with increasing concentrations of fluorescein (0 - 100 mM) shows that our instrument (Odyssey FC from LI-COR bioscience) can measure fluorescence signals between 0 and 1000 reliably.

Measurements of OD600 and fluorescence signal of E.coli cells expressing EGFP

Overall, the results showed that aeration condition affects cell growth and protein expression represented by fluorescence signal. The first group (green) with standard flask and 100 rpm resulted in the lowest fluorescence and cell growth after 120 minutes. The second group (yellow) with regular flask with 150 rpm showed a slight increase of fluorescence compared to the 1st group at every time interval. Lastly, the group with baffled flask and 150 rpm (blue) showed the highest fluorescence at every time points.

The significant figures of all measurements (fluorescence and OD600) are 3 digits. Replicate experiments were not performed due to time- and space-limitations of the laboratory.

Analysis: Higher RPM and usage of baffled flask is likely to provide higher aeration condition. Thus, we can derive from the result that higher aeration condition is optimal for EGFP expression and cell growth, particularly on the BL21 cell line.

Limitation: Because we did not directly measure the concentration of dissolved oxygen in the flask, it is not conclusive if the increased fluorescence signals and OD600 in the baffled flask are due to increased aeration. Also, the effect of RPM variations was not very evident from our experiment. To check the effect of RPM difference, we would need more trials with wider RPM ranges (50 ~ 200 rpm, for example). Also, exploring other methodology such as the use of a fermentor for more controlled aeration conditions would be beneficial to discover the exact effect of aeration on the expression of eGFP and growth of E.coli.