Team:KCIS NewTaipei/Experiments

Experiment

EXPERIMENT EXPERIMENT

PARTS
DESCRIPTION
CONFIRMATION
OF HBD & BCD
OVEREXPRESSION
ASSAYS OF
HBD & BCD
ACTIVITY
UPREGULATION
OF VDR INDUCED
BY BUTYRATE

Due to COVID-19, we couldn’t finish our wet lab since we only had one month to do the experiment. Some of the future experiments we planned can be performed if our team decides to continue on phase 2 in the next iGEM cycle.

Parts Description

*Fig.1 Gene construct for HBD and BCD gene

Our HBD was not synthesized as a composite part, therefore it was obtained directly from E.coli Nissle 1917 strain (EcN). To construct the HBD expression plasmid, primers possessing the restriction enzyme sites EcoRI and XhoI were used to amplify the HBD gene via PCR from EcN genomic DNA. The HBD gene was inserted into pET28a.

On the other hand, the BCD gene, as a composite part, was designed using Benchling. The BCD gene sequence was obtained from the genomic DNA of Faecalibacterium prausnitzii. The BCD gene, flanked by restriction sites EcoRI and SpeI, was synthesized by Twist Bioscience and was inserted into pSB1C3 via BioBrick Assembly.

*Fig.2 Insertion of our gene of interest, HBD gene and BCD gene, into our vector, pET28a and pSB1C3

We then transformed engineered pET28a and pSB1C3 into E.coli DH5𝛼 competent cell separately. The bacterial strains were cultured in Luria Broth medium, including kanamycin for pET28a, chloramphenicol for pSB1C3. After using colony PCR to test the success of transformation, the plasmids were extracted using Miniprep and inserted into BLR(DE3) competent cells since DH5𝛼 does not acquire the T7 promoter system. The culture was subsequently left to grow overnight in LB medium for protein expression assay.

*Fig.3 Inserting HBD and BCD into competent cells.

Confirmation of HBD & BCD
Overexpression

Since pET28a is a lac operon inducible system, we added IPTG into our bacterial culture to induce protein expression. We set the IPTG induced culture as the experimental group, while uninduced and culture as negative control; the wildtype culture was included in the negative control group to prevent leaky expression from the lac operon. For the BCD construct, since lac operon was not present, we set the engineered culture as the positive control, while wildtype culture as negative control.

We lysed our culture by heating the sample. Both HBD and BCD protein were purified using nickel column chromatography. The concentration of the protein was determined using a spectrophotometer at a wavelength of 280 nm.

Assays of HBD and BCD Activity

HBD

The HBD protein catalyzes the formation of 3-hydroxybutyryl-CoA from acetyl-CoA, while oxidizing NADH to NAD+.

*Fig.4 Reaction catalyzed by HBD

We added a fixed amount of acetyl-CoA and NADH, while varying the concentration of purified HBD protein. The activity of the protein was accessed through measuring the concentration of the reactant NADH, with a spectrophotometer at a wavelength of 340 nm.

BCD

We implemented the FadB-coupled assay proposed by Park et al. The BCD catalysis reaction was coupled with that of FadB, which catalyzes the formation of acetoacetyl-CoA to crotonyl-CoA, with simultaneous reduction of NAD+ to NADH.

*Fig.5 Reaction catalyzed by BCD

We added a fixed amount of butyric acid, acetyl-CoA, FAD, and NAD+, while varying the concentration of the purified BCD protein, for the reverse pathway to proceed. The activity of the protein was again accessed through measuring the concentration of the product NADH using the same method mentioned above.

Upregulation of VDR Induced by
Butyrate

In order to confirm that butyrate is able to upregulate VDR, we treated HCT cell lines with various dosages of butyrate to quantify VDR protein expression. Sodium butyrate was dissolved in PBS. The dosage of butyrate ranged from 0mM (PBS only) to 3mM, with a constant leap of 0.5mM. The HCT cell lines were cultured in DMEM. The medium of culture was replaced every day.

*Fig.6 Adding different doses of sodium butyrate into HCT cell lines.

We left HCT cells to grow in a medium containing butyrate for 48 hours. The cells were then harvested, washed in ice-cold PBS, and lysed by heating. Protease inhibitors were added to prevent protein degradation. The lysate was then sonicated, centrifuged, and quantified using Bio-Rad protein assay.

After quantification, the lysate was again analyzed using SDS-PAGE. The band at 48kDa was identified and quantified using Bio-Rad Image software. The experiment procedure was repeated three times. The datas were used for modeling efforts.