SulFind 2021

Improvement of an Existing Part

Table 1: Part overview

Name	Type	Description	Length [bp]
BBa_K3762010	Composite	YFP_LOV driven by T7 promoter	528

Our project design affected which part we ended up with and how we improved it. One of our initial project approaches included using a whole-cell sensor in a microfluidic chip that would detect H₂S in the water that moves across the sensor. In this environment, the cell would be in near hypoxic conditions which would require a signal generator that would work without reliable access to molecular oxygen so that we could detect activity via its fluorescence signaling.

We wanted to modify an existing part where the improvement was in its added utility so that it would fit the needs of future teams whose project has aspects that require it to work under hypoxic conditions or where the availability of molecular oxygen is low.

Our chosen part was the well characterized signal generator “superfolder GFP driven by T7 promoter” BBa_I746909 by Cambridge 2007 in which we replaced its sfGFP with the “heat stable fluorescent YFP_LOV protein” BBa_K3427000 from GO_Paris-Saclay’s 2020 team.

The reason for replacing sfGFP in BBa_I746909 with YFP_LOV is that GFP variants require molecular oxygen in order to cyclize into its fluorescent state [1] while LOV-based fluorescent proteins do not require molecular oxygen for it to attain its fluorescent conformation [2].

The protein sequence of the YFP_LOV that GO_Paris-Saclay’s 2020 team registered is derived from an 2019 article [3] where it was a part of a protein with a histidine kinase activity from Chloroflexus aggregans, a thermophilic phototrophic bacteria.

Improvement parts (BBa_I746909 & BBa_K3762010) were ordered synthesized from IDT as linear fragments and subsequently cloned into a pENZ004 plasmid vector made available to us by the PhotoSyn lab at NTNU using Gibson assembly, and transformed into E. coli BL21 cells.

YFP_LOV driven by T7 promoter

The pENZ004 plasmid has ampicillin resistance, and a ColE1 origin of replication. The T7 expression that this part is based on is inducible by isopropyl-β-D-thiogalactopyranoside (IPTG). The T7 expression system is also leaky, which will result in background expression of products downstream of the T7 promoter without an inducer.

Figure 1: Plasmid map of the pENZ004 plasmid and our construct BBa_k3762010.

The added functionality that BBa_K3762010 brings compared to BBa_I746909 is that it does not require molecular oxygen in order to attain its fluorescent state. BBa_K3427000

Characterization

The linear fragment of BBa_K3762010 was cloned using Gibson assembly into a pENZ004 plasmid provided by the PhotoSyn lab at NTNU. We proceeded to transform the plasmid into our E. coli BL21 cells and selected using ampicillin resistance.

Fluorescence measurement settings using a Tecan Infinite 200 Pro:

Table 2: Tecan Infinite 200 Pro settings for measuring fluorescence intensity in BBa_I746909

Setting	Value
Excitation wavelength	470 nm
Emission wavelength	520 nm
Excitation Bandwidth	9 nm
Emission Bandwidth	20
Gain	100
Number of flashes	22
Integration Time	20 us
Lag time	0 us
Settle time	0 ms
Z-position	20000 um

Table 3: Tecan Infinite 200 Pro settings for measuring fluorescence intensity in BBa_K3762010 in accordance with the characterization protocol in BBa_K3427000

Setting	Value
Excitation Wavelength	488 nm
Emission Wavelength	536 nm
Excitation Bandwidth	9 nm
Emission Bandwidth	20
Gain	100
Number of flashes	22
Integration Time	20 us
Lag time	0 us
Settle time	0 ms
Z-position	20000 um

Results

Figure 2: An edited .tiff image taken from a Bio-Rad GelDoc showing the difference in fluorescent color between the negative culture; E. coli BL21 cells and samples with our construct containing YFP_LOV under T7 control; Control, 40 µM IPTG & 400 µM IPTG.

Figure 3: Fluorescence intensity plot for four parallels of BBa_K3762010.

Table 4: Result matrix for our experiment with BBa_K3762010.

Sample	Plasmid	Inducer (IPTG)	Fluorescence
Negative	No	No	No
Control	Yes	No	Yes
40 µM IPTG	Yes	Yes	Yes
400 µM IPTG	Yes	Yes	Yes

Figure 4: Fluorescence intensity plot for two parallels of BBa_I746909. Excitation: 470 nm. Emission: 520 nm.

Table 5: Result matrix for our experiment with BBa_I746909

Sample	Plasmid	Inducer (IPTG)	Fluorescence
Negative	No	No	No
Control	Yes	No	Yes
40 µM IPTG	Yes	Yes	Yes
400 µM IPTG	Yes	Yes	Yes

Sequencing results from Eurofins confirmed successful insert of plasmids with each respective construct.

Conclusions

Sequencing confirmed successful insert in the pENZ0004 plasmid and results indicate that we were able to clone our construct into our E. coli BL21 cells.

Results indicate that BBa_K3762010 is not as fluorescent as BBa_I746909 but does exhibit similar behavior with and without inducers in our inducer concentration range [40 - 400 µM IPTG].

Due to how flavin-based fluorescent protein does not require molecular oxygen to attain its fluorescent form, the case for the improvement over sfGFP lies in its function in hypoxic conditions. Potential applications can be in microfluidics or a diagnostic setting where the environment has low or none molecular oxygen availability.

References

:

[1] Limitations of the Reporter Green Fluorescent Protein under Simulated Tumor Conditions

Claudia Coralli, Maja Cemazar, Chryso Kanthou, Gillian M. Tozer and Gabi U. Dachs

Cancer Res June 15 2001 (61) (12) 4784-4790;

https://cancerres.aacrjournals.org/content/61/12/4784

[2] The photophysics of LOV-based fluorescent proteins - new tools for cell biology

Marcus Wingen, Janko Potzkei Stephan Endres, Giorgia Casini, Christian Rupprecht, Christoph Fahlke Ulrich Krauss, Karl-Erich Jaeger, Thomas Drepper and Thomas Gensch

Photochem. Photobiol. Sci., 2014,13, 875-883

https://doi.org/10.1039/C3PP50414J

[3] A thermostable flavin-based fluorescent protein from Chloroflexus aggregans: a framework for ultra-high resolution structural studies Nazarenko et al. 2019, Photochem

Photochem. Photobiol. Sci., 2019,18, 1793-1805

https://doi.org/10.1039/c9pp00067d