SOEing:

The gBlocks for the VDR-FRET and OHase constructs were both delivered in two separate parts due to production constraints. It was therefore necessary to create the complete VDR-FRET and OHase constructs prior to cloning. The four total delivered parts were respectively termed mCherryVDR, mNeonGreenVDR, OHase1, and OHase2. To facilitate PCR SOEing, they were subjected to PCR amplification.

Figure 1. PCR Amplification of mCherryVDR, mNeonGreenVDR, OHase1,
and OHase2 gBlock constructs

From Figure 1 it is seen that amplification of the gBlocks was successful. The highest bands in the Cherry VDR and the Green VDR correspond roughly to the expected sizes of the gBlocks (1215 bp and 1191 bp respectively). Furthermore, the highest bands in the OHase1 and OHase2 lanes also correspond roughly to their expected band sizes (849 bp and 842 bp respectively). The results from this test indicated that it would be possible to use the products in the syntheses of the next step.

In order to combine the VDR-FRET and OHase sub-pieces, a technique called PCR SOEing was used. Initially, with this technique, mCherryVDR and mNeonGreenVDR were fused together to produce the whole VDR-FRET sequence.

Figure 2. PCR SOEing results of
the VDR-FRET construct (mNeonGreenVDR + mCherryVDR)

As seen in Figure 2, the highest band observed in the VDR-FRET lane corresponds to roughly 2.5 Kb, which is in concordance with the size of the VDR-FRET construct size (2 426 bp). This suggests that VDR-FRET was successfully synthesized from the mNeonGreenVDR and mCherryVDR gBlocks. Notably, there were extra bands observed at lower size values, indicating that the SOEing of VDR-FRET was not efficient.

Next, the fusion of the OHase1 and OHase2 constructs were tested, again using the PCR SOEing technique, with the goal of creating a complete OHase construct.

Figure 3. PCR SOEing results of
the OHase construct (OHase1 + OHase2)

In Figure 3 there is a bright band observed at around 1.8 Kb, which corresponds roughly to the size of the OHase construct (1 757 bp). This indicates that there was successful amplification and synthesis of the OHase DNA. Although, similarly to the VDR-FRET SOEing results (Fig. 2), there were extra bands observed.

Cloning/Transformation:

Following the successful creation of VDR-FRET and OHase, the constructs were ready to be introduced into an E. coli system. First, VDR-FRET was ligated into the pSB1C3 plasmid to form a plasmid we named pGEM26 (Fig. 5). This plasmid was then transformed into DH5α E. coli cells, which were grown on selective chloramphenicol media. Colonies that formed were subjected to colony PCR testing using the end primers of the VDR-FRET construct.

Figure 4. Colony PCR results of transformation
of VDR-FRET into pSB1C3.

Put Image 6
Figure 5. Plasmid map of pGEM26, constructed by the
BioBrick insertion of the VDR-FRET sequence into pSB1C3.

The agarose gel in Figure 4 shows that for each colony selected, following PCR amplification of VDR-FRET, there were observed bands around 2.4 Kb. This suggested that there was successful ligation of VDR-FRET into the pSB1C3 plasmid as well as successful transformation into E. coli.

Next, the OHase construct was ligated into the previously produced pGEM26 plasmid to produce a plasmid we named pGEM27 (Fig. 7) and transformed into E. coli. Two colonies were selected from the overnight growth of the transformed cells, and were subject to PCR amplification using primers designed for the sequencing of OHase, as well as VDR-FRET (to verify the presence of both parts).

Figure 6. Colony PCR results of transformation
of OHase into pGEM26.

In Figure 6, we used primers that amplified segments of each sequence; the bands were at the expected sizes of the segments (536 bp of the OHase sequence, and 833 bp of the VDR-FRET sequence).

Put Image 7
Figure 7. Plasmid map of pGEM27, constructed by
the BioBrick insertion of the OHase sequence into pGEM26.

Sequencing:

From the results in Figure 4, colonies 2.1 and 2.2 were selected to be subject to DNA sequencing in order to further ensure the proper incorporation of the VDR-FRET construct. From the results in Figure 5, the OHase colony was selected to be subject to DNA sequencing in order to ensure the proper incorporation of the OHase construct.

Figure 8. Sequencing results of the VDR-FRET transformation in colony 2.1

Figure 8 shows that the VDR-FRET construct for colony 2.1 was mostly sequenced and found to contain little error. The only notable mistake in this construct is the conversion of amino acid 72 from a histidine to an arginine. Despite this, there are also a few regions of the VDR construct that were not sequenced due to the lack of primer sequencing overlap. There is missing sequencing data for a 46 bp length in the mNeonGreen fluorophore region as well as a missing 50 bp in the VDR-LBD region.

Figure 9. Sequencing results of the VDR-FRET transformation in Colony 2.2

Figure 9 shows that sequencing results for colony 2.2 showed more errors than for colony 2.1. The main nucleotide substitution errors consisted of:

A → T change (Ile → Phe) in the mCherry region
T → C silent mutation in the VDR region
C → A change (Pro → Gly) in the VDR region
G → T change (Glu → stop) in the mCherry region
C → T change (Arg → Gly) in the mCherry region

There was also a deletion in the VDR-LBD part of the sequence that would have had downstream effects. Also to be noted, there is an approximately 600 bp gap region of unknown sequence at the end of the mCherry region, including the BioBrick suffix.

Figure 10. Sequencing results of the OHase transformation into pSB1C3_VDR-FRET

Figure 10 shows the results for the colony containing the OHase construct within its pSB1C3 plasmid. From these results, a few mistakes were observed:

C → T change (Gly → Val)
C → A silent mutation
Deletion of A at 1 439

Assay 1:

After the confirmation of successfully transformed cells, the next step was to measure the fluorescence of our system. To develop a fluorescence baseline, the fluorescence of the system without vitamin D was tested. These tests were performed in triplicate for induced and uninduced E. coli BL21 cells as well as untransformed BL21 cells.

Figure 11. mNeonGreen fluorescence (RFU) of induced, uninduced,
and untransformed BL21 cells as a function of μg of protein from crude lysate, measured with
AutoGain.

From Figure 11 it was observed that by increasing the amount of crude lysate, the fluorescence of mNeonGreen after being exposed to light at a wavelength of 485 nm increased proportionally. This was the case for induced cells, uninduced cells, and untransformed BL21 cells. The highest response was found with the uninduced cells expressing VDR-FRET and the lowest was found with the cells that were induced and expressing VDR-FRET.

Next, the fluorescence of mCherry was tested for transformed cell lysates against untransformed BL21 cell lysate by light excitation at a wavelength of 540 nm and emission measurement at a wavelength of 610 nm.

Figure 12. mCherry fluorescence (RFU) of induced, uninduced,
and untransformed BL21 cells as a function of μg of protein from crude lysate, measured with
AutoGain.

From Figure 12 it is seen that both the induced and uninduced cells expressing VDR-FRET displayed an increase in mCherry fluorescence response compared to normal BL21 cells, which did not display any increase. Although, the degree to which the induced and uninduced cells responded with fluorescence is similar.

Lastly, the mNeonGreen/mCherry FRET system as a whole was tested for the uninduced, induced, and untransformed BL21 cells by excitation with light at a wavelength of 485 nm and measuring the emission response at a wavelength of 620 nm, with the addition of increasing amounts of lysates.

Figure 13. FRET fluorescence (RFU) of induced transformed, uninduced transformed, and
untransformed BL21 cells as a function of μg of protein from crude lysate, measured at gain = 110.

From Figure 13 it can be seen that the induced and uninduced cell lysates respond to mNeonGreen excitation wavelengths and respond with mCherry emission wavelengths to high degrees, in comparison to BL21 cell lysate. Similarly to the mCherry testing in Fig. 12, the responses of the induced and uninduced cell lysate product have little variability in comparison to each other.

Assay 2:

The next step was to determine the range to which certain concentrations of vitamin D affect the observed FRET fluorescence, reflecting on relevant biological levels. The selected concentrations of vitamin D to test were 20 nM and 60 nM

Initially, increasing amounts of induced and uninduced cell lysates had vitamin D added to a final concentration of 20 nM. These reaction volumes were excited to normal FRET excitation (485 nm) and emission (620 nm) wavelengths.

Figure 14. FRET fluorescence (RFU) of induced transformed cells at final [1,25(OH)₂D] of 20 nM,
as a function of μg of protein from crude lysate, measured with AutoGain.

Figure 15. FRET fluorescence (RFU) of uninduced transformed cells at final [1,25(OH)₂D] of 20 nM,
as a function of μg of protein from crude lysate, measured with AutoGain.

Following this, the same conditions of increasing induced and uninduced cell lysate were tested in reaction volumes with final vitamin D concentrations of 60 nM.

Figure 16. FRET fluorescence (RFU) of induced transformed cells at final [1,25(OH)₂D] of 60 nM,
as a function of μg of protein from crude lysate, measured with AutoGain.

Figure 17. FRET fluorescence (RFU) of uninduced transformed cells at final [1,25(OH)₂D] of 60 nM,
as a function of μg of protein from crude lysate, measured with AutoGain.

From Figures 14-17 it can be seen that the fluorescence response increases alongside the addition of both 20 nM and 60 nM of vitamin D. Observed in each graph were degrees of variability across the triplicate data. It was found that the least amount of variability in fluorescence response was found when using 60 µg of cell lysate.

Assay 3:

The next test was to find out if our induced and uninduced cell lysates, as well as if our untransformed BL21 cell lysates, respond with fluorescence to concentrations of vitamin D ranging from 10 nM to 250 nM. Data taken from assay 2 prompted the use of 60 µg of lysate for this test.

Figure 18. Relative FRET fluorescence of 60 μg protein from crude lysates of induced, uninduced,
and untransformed BL21(DE3) cells with pSB1C3_VDR-FRET upon addition of 1,25-dihydroxyvitamin D at gain = 110.

From Figure 18 it can be seen that there are slight increases to the fluorescence responses of the induced, uninduced, and BL21 cell lysates. The largest increases in fluorescence are observed with the induced cell lysates, followed by the uninduced cell lysates. The lowest increases in fluorescence were observed with the BL21 cell lysates.

Protein Purification:

Cell lysates of the VDR-FRET induced, uninduced and untransformed BL21 cells were purified using NEBExpress® Ni Spin Columns (NEB #S1427) and viewed on an SDS-PAGE gel, alongside their unpurified counterparts.

Figure 19. SDS-PAGE of crude and purified lysates of induced, uninduced,
and untransformed BL21(DE3) strains.

In Figure 19 it is shown that His-Tag purification of VDR-FRET involved the copurification of numerous other proteins. This is shown by the relative distribution of protein bands observed across crude lysate protein as well as purified protein and supernatant. There is a unique band observed in the induced purified supernatant at roughly 80 kDa, similar to the expected band size of VDR-FRET (83 kDa). Similar protein band intensities were observed for each lane, except for the induced purified protein lane, which displayed less overall bands and band intensities.