Part:BBa_K4939000
vsfGFP-0
An engineered variant of sfGFP, which has shown enhanced stability and fluorescence in in vitro and in vivo models.
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal EcoRI site found at 664
Illegal PstI site found at 931 - 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 664
Illegal PstI site found at 931 - 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 664
- 23INCOMPATIBLE WITH RFC[23]Illegal EcoRI site found at 664
Illegal PstI site found at 931 - 25INCOMPATIBLE WITH RFC[25]Illegal EcoRI site found at 664
Illegal PstI site found at 931
Illegal NgoMIV site found at 835
Illegal AgeI site found at 573 - 1000COMPATIBLE WITH RFC[1000]
Description
It has been widely reported that the binding of certain single domain antibodies (nanobodies) allows for the stabilisation of GFPs (which can be inherently unstable), and thus enhances their fluorescence. One particular nanobody - the enhancer nanobody - was shown to significantly increase fluorescence when bound to GFP. Eshagi et. al[1], reasoned that directly fusing this nanobody to sfGFP could create a novel GFP with enhanced fluorescence, since the stabilisation was now intramolecular and thus built into the fold of the protein. Indeed, they were correct and the newly designed vsfGFP-0 showed ~3 times more fluorescence than sfGFP. Notably, the group developed two versions: vsfGFP-0, in which the fusion of the enhancer nanobody gave rise to a dimerisation prone GFP; and vsfGFP-9 in which a 9aa linker was utilised between the domains which promoted monomeric folding - the former (i.e., the dimerised variant) is the one discussed here.
Part Improvement
This GFP corresponds to the second brightest in the Fluorescent Protein database. For this reason, and due to its potential increased functionality as a brighter daughter molecule to the well used and characterised sfGFP, the iGEM Sheffield team took on the challenge to compare the fluorescence between the two related proteins. We did this by cloning vsfGFP-0 and sfGFP into pET28a vectors and inducing Lemo BL21 E. coli transformed with these constructs to express the proteins. The pET28a vector contains by default the Lac, IPTG-inducible mechanism, which was harnessed to obtain different levels of gene expression depending on the concentration of inducer.
Experimental Design
The experiments were conducted in 96 well plates and measured using a Hidex Sense Microplate Reader Type 425-311 (g/n 320-0250). There were 150uL of culture in each well in total. 130uL of these were made up of 2.5% Millers LB media with kanamycin (50ng/uL) and chloramphenicol (25ng/uL). The remaining 20uL were made of the relevant culture diluted to an OD of 0.05. A culture transformed with a construct comprising vsfGFP-0 expressed by the Anderson promoter J23110 was added as a positive fluorescence control; and a culture transformed with the original "empty" pET28a vector was also added as a positive growth control. The arrangement of the plate is shown in Figure 1.
Figure 1. Shows the spatial arrangement of cultures and the relevant concentration of IPTG they were induced by. Assume appropriate concentration of antibiotics.
Results and Analysis
All inducible constructs showed an increase in fluorescence as the concentration of IPTG increased, although those from rows A and B had a much steeper increase than those in rows C and D, as shown in Figure 2. This suggests an intrinsic superiority in the brightness of the protein expressed by the bacteria. The growth control, as expected, showed near-zero levels of fluorescence and the fluorescence control saturated the sensor. Well D1, which was meant to be a blank, was contaminated when loading the plate, which matches the unexpected growth shown by the measurements.
Figure 2. Shows a visual representation of the behaviour of OD with respect to time (h) in each well of the plate.
When formatting the plots depicting fluorescence with respect to time, it is very easy to see that for every sfGFP construct at a particular IPTG concentration there is a vsfGFP-0 analogous that exhibits a higher fluorescence corresponding to the same inducer concentration every time. See Figure 3 where the fluorescence values have been corrected against the fluorescence of the blank.
Figure 3. Shows mean corrected fluorescence (RFU) measurements with respect to time (h) for different concentrations of IPTG in cultures transformed with vsfGFP-0 in pET28a, and a positive fluorescence control under the control of the J23110 Anderson promoter is also included.
Processing the data further gives some more interesting insight. If the mean values of fluorescence at a given time are divided by the optical density at that time, the quotient will be a magnitude representing "fluorescence per cell". This is the most important metric. Since GFPs (and in particular sfGFPs) are so stable, it is necessary to obtain a normalised value for fluorescence, as in time the GFPs will build up and the values of fluorescence will become an indicator of how long the bacteria have been expressing the protein rather than how fluorescent the protein actually is.
In Figure 4 it can, once more, be clearly seen that the F/OD increases as the inducer concentration increases and the vsfGFP-0 always leads in magnitude with respect to its sfGFP analogous when both are subjected to the same IPTG concentrations.
Moreover, it is important to note the trough in F/OD at approximately four hours. This might indicate an offset between the times when the logarithmic phase begins for growth rate and when it begins for GFP production, or it could also denote the required maturation time for GFPs to begin fluorescing. It seems as though cells begin dividing rapidly roughly five hours before they begin producing protein, and are subsequently surpassed by a rapid increase in GFP production. This would explain the decrease in the F/OD quotient from when measurements started to be recorded until around five hours later.
Figure 4. Shows fluorescence per cell (RFU/OD) measurements for different concentrations of IPTG in cultures transformed with vsfGFP-0 in pET28a, and a positive fluorescence control under the control of the J23110 Anderson promoter is also included.
Finally, utilising all the data points measuring fluorescence per IPTG concentration we can plot the trend followed by the cultures, shown in Figure 5. This indicates that the fluorescence of the cultures with each of the reporters tested increases with the concentration of inducer. However, vsfGFP-0 increases significantly faster than sfGFP establishing, indicating considerably higher brightness. At the highest IPTG concentration, overall, the measured fluorescence of the culture containing the vsfGFP-0 gene was 2.5 times more fluorescent than that of sfGFP.
It should be highlighted that these trendlines do not cross the origin. Additional to potential systematic errors in measuring, this could depict leaky expression of the T7-pLac which manages the expression of the GFP gene, as has been previously described in literature[2]. The error bars were measured based on the standard error of mean of each point. It should be noted that mean was done between the corresponding plasmids (A and B or B and C). Moreover, as seen at 3.5μΜ, the error bars overlap. This happens because the two values have a range higher than 500,000 RFU OD-1. In addition, these relationships could be used further to predict relative maximum F/OD depending on the concentration of inducer. This shall be explored further.
Figure 5. Shows the trends in fluorescence (RFU) per IPTG concentration (uM) for cultures transformed with vsfGFP-0 in pET28a, sfGFP in pET28a and pET28a with no insert.
Conclusions
It seems reasonably clear the improvement vsfGFP-0 presents over its parent molecule sfGFP as a reporter, as equivalent gene expression results in significantly higher fluorescence in the former.
Figure 6. Depicts the case where the concentration of IPTG is 5uM, and draws a clear representation of the conclusion that has been arrived to.
Figure 7. Allows for a visual comparison between brightness of the two cultures transformed with the two different fluorescent reporters.
References
[1] M. Eshaghi et al., “Rational Structure-Based Design of Bright GFP-Based Complexes with Tunable Dimerization,” Angewandte Chemie (International ed.), vol. 54, no. 47, pp. 13952–13956, 2015, doi: 10.1002/anie.201506686.
[2] F. Du et al., “Regulating the T7 RNA polymerase expression in E. coli BL21 (DE3) to provide more host options for recombinant protein production,” Microbial Cell Factories, vol. 20, no. 1, 2021. doi:10.1186/s12934-021-01680-6
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