Difference between revisions of "Part:BBa S03452"

Revision as of 06:39, 17 October 2019

pCX-EGFP

enhanced green fluorescent protein.

Usage and Biology

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

Sequence and Features

Measurements by KORHS team

Aim: For our characterization experiment, our team has chosed to characterize the part BBA_S03452. The iGEM registered part BBA_S03452, EGFP(Enhanced green fluorescence protein) is a type of fluorescent protein whose fluorescence measurement can be used to quantify protein. Our team used the cell line BL21(DE3) for this project and 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 and protein expression.

Method : We divided three Experimental Group 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) 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 for all time.

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 represents 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 dues 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 use of a fermentor for more controlled aeration change would be beneficial to discover the exact effect of aeration on the expression of eGFP and growth of E.coli.