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Experiments & Results

Technology roadmap

Content

1. SNP Treatment

2. sGC Overexpressing Device

3. PDE5A Silencing Device

4. Synergetic effects of sGC Overexpressing and PDE5A Silencing Devices

5. Hypoxia Responsive Promotor

1.SNP Treatment

In the preliminary experiments, we treated HEK 293 cells with 10umol/L sodium nitroprussiate (SNP), a NO donor, for different time periods. Cellular concentrations of cGMP were examined using a commercial Elisa Kit. As shown below, cellular concentration of cGMP increased after SNP treatment and peaked at 20 minutes. We used this condition as a positive control in our later experiments.

Fig.1 cGMP concentration after treated with 10umol/L SNP

2.sGC Overexpression

Construction:

Lentivirus vectors carrying the α or β subunits of sGC were created using the scarless golden gate assembly. The sequence encoding a fluorescent reporter was inserted after the antibiotics-resistance gene, rather than directly after the alpha3 and beta3 gene, to avoid any potential influences on the function of the part.

Vector Map of Alpha3 subunit

Fig.2 Lentivirus vector cointaing guanylate cyclase 1 alpha3 subunit gene

Vector Map of Bata3 subunit

Fig.3 Lentivirus vector cointaing guanylate cyclase 1 beta3 subunit gene

Confirmation of DNA Sequence：

We confirmed the construction of hGUY1A3 gene and hGUY1B3 gene encoding the α and β subunits of sGC, respectively, into Biobrick format flanking by required restriction sites (BBa_K1720000 and BBa_K1720001).

Transfection：

HEK293 cells were transfected with the designed devices. Vectors carrying α subunit of sGC was inserted with mCherry gene as a reporter (red) while vectors carrying β subunit of sGC was inserted with EGFP gene as a reporter (green). Both red and green fluorescence were observed under fluorescence microscope. The transfection appeared to be successful!

Fig.4 Red fluorescence signal after transfection of alpha3 subunit

Fig.5 Green fluorescence signal after transfection of beta3 subunit

Gene expression levels detection：

To examine gene expression of the key players in the cGMP metabolism pathway, we designed primers for hGUY1A3, hGUY1B3 and PDE5A. The house keeping gene GAPDH was used as an internal control. Results of RT-PCR and Real-time PCR were shown below. After transfection of the α and β subunits of sGC, the expression levels of α and β subunits were up-regulated. But the expression of PDE5A did not change significantly.

Fig.6 Transcription level after transfection with alpha3 and beta3 subunit

Fig.7  △CT vs GAPDH after transfection of alpha3 and beta3 subunit

Mesurement of sGC activity and cGMP concentration:

We next examined whether the overexpressed α and β subunits of sGC are functional. We used Elisa Kit to measure the sGC activity and cellular cGMP levels. Compared to empty vectors, transfection of the α and β subunits of sGC significantly increased sGC activity and cGMP level in HEK293 cells. Our sGC overexpressing devices worked successfully.

Fig.8 sGC activity after cotransfection of alpha and beta subunit

The result in Fig 8 indicated that sGC overexpression was successful! And we we used cGMP Elisa Kit to detect whether cGMP concentrantion increase at the same time.

Fig.9 cGMP concentration after cotransfection of alpha and beta subunit

3.PDE5A Silencing Device

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Construction:

Lentivirus vectors carrying three different PDE5A shRNA designs were created using the scarless golden gate assembly. The sequence to encode a EGFP fluorescent reporter was inserted after the Puro antibiotics-resistance gene， rather than directly after the shRNA gene, to avoid any potential influences on the function of the part.

Vector Map

Fig.10 Lentivirus vector cointaing PDE5a silencing device

Confirmation of DNA Sequence：

We confirmed the construction of shRNA gene, respectively, into Biobrick format flanking by required restriction sites (BBa_K1720003,BBa_K1720004 and BBa_K1720005).

Measurement of Gene Expression：

We used RT-PCR to see whether expression of the PDE5A gene was suppressed by our shRNA devices.

Fig.11 PED5a transcription level after transfection with three different shRNA

The results showed that the expression of PDE5A was silenced by all three shRNA devices.

Measurment of cGMP level:

Fig.12 cGMP concentration after treated with three different shRNA

When we measured cellular cGMP levels, shRNA3 showed better potential. Therefore, we chose shRNA3 as our best PDE5A silencing device for further study (part number: BBa_K1720005).

4.sGC and shRNA cotransfection

Measurment of cGMP level:

In the next step, sGC Overexpressing and PDE5A Silencing Devices were co-transfected into HEK293 cells. Again, we used Elisa kits to detect the sGC activity and cellular cGMP levels. Whereas either device functioned well by themselves, a synergetic effect between the sGC overexpressing device and the PDE5A silencing device was also observed!

Fig.13 sGC activity after transfection of sGC vectors and shRNA vectors

We used cGMP Elisa kit to detect the cGMP concentration to see whether cGMP concentration will be up regulated after cotransfection.

Fig.14 cGMP concentration after cotransfection of sGC vectors and shRNA vectors

5.Hypoxia Responsive Promotor

Construction:

We also designed a hypoxia responsive CMV promotor by inserting a hypoxia responsive element （HRE） to the CMV promoter,BBa_k747096, submitted by the team Freiburg in 2012.

Vector Map

Fig.15 Vecror containing hypoxia responsive CMV promotor and EGFP reporter

Confirmation of DNA sequence:

We confirmed the construction of hypoxi responsive promotor into Biobrick format flanking by required restriction sites (BBa_K1720002).

Transfection：

HEK293 cells were transiently transfected with the device, which carried the hypoxia-induced promotor followed by an EGFP florescent reporter (green). The cells were either cultured under normoxia situation or treated with sodium hyposulfite, an oxygen cleaner to cause hypoxia situation, for 2 hours. As a control, HEK293 cells were also transiently transfected with carrying the original CMV promoter, submitted by the team Freiburg in 2012, followed by the EGFP reporter.

Fig.16 EGFP signal under the control of CMV promotor

Fig.17 EGFP signal under the regulate of HRE in normoxia situation

Fig.18 EGFP signal under the regulate of HRE in hypoxia situation

Unfortunately, we could observe  green fluorescence under the control of hypoxia responsive promotor even stronger under normoxia situation. That was mean this device could not work as we expected. After discussion,we thought there were several issues leading wrong results,such as the bad growth of cells or weaker activity of EGFP under hypoxia situation. In order to reduce the influence of oxygen activity of EGFP,we measured the transcription of hypoxia responsive promotor we designed and normal CMV promoter under normoxia/hypoxia situation with RT-PCR.

Fig.19 The transcription of hypoxia responsive promotor and CMV promoter under normoxia/hypoxia situation.

From the results,we can see that the transcription of hypoxia responsive promotor is more than CMV under hypoxia situation,and less under normoxia situation.That proved the hypoxia responsive promotor could work as we expect,though it could not work much better.We believe we can improve this promotor and make it more sensitive in our future work.