Team:Lund/Results/GFPProduction
Results
Absorbance measurements for VHb
The VHb absorbance characteristics were observed using spectrophotometry. As the role of VHb is that of an an oxygen carrier, the spectra in its oxidized, oxygenated and reduced states can be recorded in the visible range. In its oxidized state, the absolute spectrum for the initial state of the VHb is 576, 543, and 414 nm while the oxidized and reduced states have a shift around the 414 nm peak [2] as is shown by fig. 1.
The results shown in fig. 1 was from expression of all VHb constructs with 25% media and 75% headspace. From the left curve, it can be seen that all the constructs show the expected spectrum at the oxygenated state. Hence, it is shown that VHb is expressed at all the promoter strengths. Moreover, the curve also shows a different absorbance for each construct with different promoter strengths. Furthermore, it was also necessary to show the functionality of the VHb. For this, we picked BBa_K2602013 to represent the different oxidizing level as its absorbance was the highest when compared to the other constructs. Thus, very small amounts of sodium dithionite (NaD) was added into the cuvette after taking the first measurement. The NaD reduces the iron in the heme group of the VHb and affects the spectrum from 414 nm to around 425 nm as it is shown by the right curve in fig. 1 [2]. Moreover, the oxidized state was observed when the reduced VHb was bubbled with CO for around 5 seconds. Consequently, the maximum spectrum was shifted from 425 nm to 420 nm. In conclusion, the VHb constructs are active.
Measurements for VHb-GFP
Fig. 5 proved that both proteins were co-expressed by E. coli BL21 with 27 kDa for the GFP and 15.8 kDa for VHb. In order to observe the influence on VHb-GFP co-expression, different culture:headspace ratios were tried. Initially, we tried with 25% LB media in 2 L baffled flasks. The identification of GFP spectra was observed by applying the wavelength range at 350 to 650 nm, as is shown in fig. 2.
As can be seen from fig. 3, the GFP spectrum peak is observed at 504 nm. All the constructs with various promoter strengths show this peak. Thus, the GFP expression is also confirmed under the visible spectrum.
The concentration of GFP was determined by using the molar extinction coefficient E504= 5 cm-1 mM-1 [1]. The highest peak closest to λ=509 nm was chosen and converted into molar concentration. Furthermore, the character of the peak was also observed as previously described [2][3].
The GFP content of each construct at different culture:headspace ratios were plotted against the relative strength of the VHb promoter used in the biobrick. A simple linear regression was done to estimate if there is a positive correlation between the production of GFP and the VHb:s promoter strength. The slope of the linearization was estimated along with their 95% two-sided confidence intervals (Table 1, fig. 4). By looking at the intervals of the slope it can be clearly seen that the promoter strength on VHb has a significant positive effect on the level of expression of GFP, especially at low media levels. This may be due to the fact that GFP is a highly oxygen-dependent protein and the cultivations with low media levels (25%) have greater surface area that allows a higher mass transport of oxygen which maximizes the effect of VHb on GFP production at high cell density levels.
On the other, the statistical analysis of the assay performed with low media level and a gas phase enriched in carbon monoxide, fig. 4, show that carbon monoxide (CO) has a negative effect on the VHb as it has a higher diffusion coefficient and a stronger affinity to the hemoglobin compared to oxygen. Therefore, these assays indicate that VHb and its level of expression have a positive effect on the production of GFP; and possibly other recombinant proteins, in aerobic and microaerobic environments with the presence of inhibitors like CO at low concentrations.
References
[1] Bizzarri, R., Nifosì, R., Abbruzzetti, S., Rocchia, W., Guidi, S., Arosio, D., Garau, G., Campanini, B., Grandi, E., Ricci, F., Viappiani, C. and Beltram, F. (2007). Green Fluorescent Protein Ground States: The Influence of a Second Protonation Site near the Chromophore†,‡. Biochemistry, 46(18), pp.5494-5504.
[2] Chung Y. Liu and Dale A. Webster. (1973). Spectral Characteristics and Interconversions of the Reduced, Oxidized, and Oxygenated Forms of Purified Cytochrome o*. The Journal of Biochemical Chemistry, 240(13), pp. 4261-4266
[3] Jung, G., Wiehler, J. and Zumbusch, A. (2005). The Photophysics of Green Fluorescent Protein: Influence of the Key Amino Acids at Positions 65, 203, and 222. Biophysical Journal, 88(3), pp.1932-1947.
