Difference between revisions of "Part:BBa K1720000"

Revision as of 12:02, 5 September 2015

Human guanylate cyclase1,soluble, alpha 3 unit

This is a part used for coding soluble human guanylate cyclase subunit:Alpha 3.Alpha 3 unit interacts with a beta subunit to form the guanylate cyclase enzyme. Soluble guanylate cyclases(sGC) are heterodimeric proteins that catalyze the conversion of GTP to 3',5'-cyclic GMP and pyrophosphate. It is important for smooth muscle relaxation in the cardiovascular system. sGC contains an HNOX domain, which serves as a receptor for ligands such as nitric oxide, oxygen and nitrovasodilator drugs.There are several different isoforms of sGC subunit ,but the cardiovascular system abundant with alpha 3 unit and beta 3 unit.

We used lentiviral vector to transfected alpha 3 unit and beta 3 unit to HEK293 cells together with a mCherry reporter and vivo red fluorescence signal was observed under fluorescence microscope, it meant that this part was transfected into HEK293 cells successfully. Then we used qRT-PCR to observe transcriptional level of alpha 3 unit. Since soluble guanylate cyclases (sGC) are heterodimeric proteins that catalyze the conversion of GTP to 3',5'-cyclic GMP(cGMP) and pyrophosphate. The level of cGMP will be up regulated.e detected the cGMP concentration with Elisa Kit.The positive control was HEK293 cells that treat with Sodium Nitroprusside ,a NO donator that activate sGC and up regulate the level of cGMP. A negative control was made by transfecting an empty vector that does not contain sGC subunit. We used Elisa Kit to detect cGMP level. The results are as follow:

Sequence and Features

Before we start the experiment, we designed an lentiviral vector that contains our part. and packaged the vector with lentivirus.

Vector Map：

Vector Components:

alt text

Virus Titer: (3.23±2)×10^8 TU/mL

Funtional titer is determined based on q-PCR amplification of a small fragment from the lentiviral vector-WRPE that is integrated into the genome of transduced 293T cells.

Experiment 1:

At the beginning of our experiment, we aimed to prove that HEK293 cells can be transfected by our vector. In our vector we inserted mCherry gene as a repoter.Once HEK293 cells are transfected successfully red fluorescence signal will be observed under fluorescence microscope.

Protocol：

1. Seed cells to be 40% confluent at a 35mm culture dish.

2. Dilute 10ul lentiviral vector in 1ml DMEM medium containing 10% FBS

3. Withdraw culture medium from 35mm culture dish.

4. Add vector-DMEM complex to cells

5. Incubate for 15 hours.

6. Withdraw vector-DMEM complex from culture dish.

7. Add 2ml DMEM medium containing 10% FBS to cells and incubate for 10 hours

8. Observe the cells under Inverted fluorescence microscope.

Result:

From the picture we can see that vivo red fluorescence signal was observed which indicated that HEK293 cells had been transfected successfully!At the same time we also transfected HEK293 cells with vectors that contain beta3 subunit and we get the same resultS.

Experiment 2:

After we proved that HEK293 cells can be transfected, sGC alpha3 subunit gene expression levels were determined by real-time PCR.

Protocol：

Result:

Experiment 3:

As we up-regulate the transcriptional level of sGC alpha3 subunit and bata3 subunit, we used sGC Elisa kit to detect the sGC concentration to see whether two subunits combine with each other successfully.

Protocol:

1. Prepare all standards and samples be added in duplicate to the micro elisa stripplate.

2. Add standard : Set Standard wells , testing sample wells. Add standard 50 μl to standard well .

3. Add Sample : Add testing sample 10 μl then add Sample Diluent 40 μl to testing sample well(samples were 5 times diluted )Blank well doesn’t add anyting.

4. Add 100 μl of HRP-conjugate reagent to each well , cover with an adhesive strip and incubate for 60 minutes at 37°C .

5. Aspirate each well and wash by filling each well with Wash Solution (400μl ), repeating the process four times for a total of five washes. After the last wash, remove any remaining Wash Solution by decanting. Invert the plate and blot it against cleanpaper towels.

6. Add chromogen solution A 50μl and chromogen solution B 50μl to each well.Gently mix and incubate for 15 minutes at 37 ℃ Protect from light .

7. Add 50μl Stop Solution to each well.

8. Read the Optical Density ( O . D .) at 450 nm using a Microplate Reader.

Note:

1.Standard ( S0 → S5 ) concentration was followed by: 0,30,60,120,240,480 U/L

Result:

Experiment 4:

As we proved that sGC concentration was up regulate, we used cGMP Elisa kit to detect the cGMP level.

protocol: 1. Prepare all standards and samples be added in duplicate to the micro elisa stripplate.

2. Add standard : Set Standard wells , testing sample wells. Add standard 50 μl to standard well .

3. Add Sample : Add testing sample 10 μl then add Sample Diluent 40 μl to testing sample well (samples were 5 times diluted ) ; Blank well doesn’t add anyting.

4. Add 100 μl of HRP-conjugate reagent to each well , cover with an adhesive strip and incubate for 60 minutes at 37°C .

5. Aspirate each well and wash by filling each well with Wash Solution (400μl ), repeating the process four times for a total of five washes.

6. After the last wash, remove any remaining Wash Solution by decanting. Invert the plate and blot it against clean paper towels.

7. Add chromogen solution A 50μl and chromogen solution B 50μl to each well.Gently mix and incubate for 15 minutes at 37 C . Protect from light .

8. Add 50μl Stop Solution to each well.

9. Read the Optical Density ( O.D ) at 450 nm using a Microplate Reader.

Note: 1.Standard ( S0 → S5 ) concentration was followed by: 0,2,4,8,16,32 nmol/L.

Result: