Team:Hong Kong HKUST/Project/module2

Linkage

Recently, UCLA research group has introduced glyoxylate shunt to mammalian liver cell to investigate fatty acid metabolism. In their project, a constitutive promoter was used. In addition to the constitutive glyoxylate shunt, fatty acid sensing mechanism team is trying to introduce an inducible system that allows tunable fatty acid uptake by sensing fatty acid concentrations. It prevents risk of fatty acid deficiency at a low fatty acid concentration.

Sensing Mechanism

Mechanism of fatty acid metabolism regulator protein (FadR)

Fatty acid metabolism regular protein (FadR) is a bacterial transcription factor that regulates lipid metabolism of fatty acid biosynthesis and beta-oxidation. The binding of fadR is inhibited by fatty acyl-CoA compounds, which are intermediates of fatty acid degradation. HKUST iGEM group has designed to use this protein in mammalian cell to sense the amount of fatty acid present in the cell.
In the absence of fatty acid, a constitutively expressed fatty acid metabolism regulator protein FadR binds to Pfad promoter (pFadBA) and inhibits the expression of aceA and aceB proteins. In our project, we aim to use this sensing mechanism to regulate the transcription of aceA and aceB genes for glyoxylate shunt. As a regulatory system, when fatty acid is introduced to HepG2 cell, fatty acid is converted into acyl-CoA, which binds to fadR and inhibits repression of Pfad.
In terms of promoter efficiency, the difference in prokaryotic and eukaryotic transcription mechanisms gives this protein low possibility to be expressed in mammalian cell. However, HKUST group plans to investigate the efficiency of prokaryotic transcription factor in eukaryotic system and compare efficiency with other sensing mechanisms, which are believed to be present in mammalian cell.

Mechanism of Binding Immunoglobulin Protein, HSPA5 or Glucose Regulated Protein (GRP78)

GRP78 (HSPA5) is involved in the folding and assembly of proteins in the endoplasmic reticulum (ER). The level of GRP78 is believed to be strongly correlated with the amount of secretory proteins (e.g. IgG) within the ER, which suggests its key role in monitoring protein transport through the cell.
High concentration of fatty acids disrupts cell homeostasis, causing endoplasmic reticulum stress (ERS) activating the unfolded protein response (UPR) that consists of 3 transmembrane proteins: IRE1 PERK and ATF6. Three signals constitutively activate the GRP78 promoter with the help of other factors, such as NF-Y, ERSF, YY1 and cleaved ATF6, acquired from the normal stress response followed by UPR.

Mechanism of Fatty Acid Binding Protein (FABP)

Fatty acid binding proteins (FABPs) are lipid-binding proteins that regulate fatty acid uptake and transfer between extra-and intracellular membranes. There are 9 different FABPs identified with tissue-specific distribution, including FABP1 in liver. With similar structure of all FABPs,the FABP genes consist of 3 introns and 4 exons.
It is believed that the FABP1 is regulated in response to fatty acid level inside the cell through two mechanisms: 1) Fatty acid in liver cell binds to the PPAR and up-regulates the expression of Fral, the enhancer of FABP gene. 2) Fatty acid stabilizes Fral mRNA and FABP mRNA and increases expression. While the regulation factors of two mechanisms remain unclear, activity of FABP promoter is believed to be corresponding to fatty acid concentration.

Experiment Flow

In investigating the promoters, we characterized them by fusing the promoters with a reporter, Green Fluorescent Protein (GFP), which we cloned from pEGFP-N1 plasmid and place it in mammalian expression vectors , BBa_J176171 and pEGFP-N1.

FadR and FadBA

In terms of FadR protein, we obtained it from 2013 iGEM DNA distribution kit part, known as BBa_K817001. We placed FadR in a mammalian expression vector, BBa_J176171. However, since FadR is a bacterial protein, we did a construction that includes a Kozak sequence as the initiation of translation process in eukaryotic cell and NLS (Nuclear Localisation Sequence) to make it be able to go back to nucleus and regulate FadBA promoter. Kozak sequence and NLS are originally found in the plasmid BBA_J176171, therefore we ligated fadR to J176171 between Kozak and NLS. Followed by restriction digestion of the protein including Kozak sequence, FadR and NLS, we put the construction to pEGFP-N1 plasmid and cut out the eGFP since eGFP is not needed in this part.
So does for FadBA promoter, we obtained it from 2013 iGEM DNA distribution kit, known as BBa_K817002. We extracted it and placed it in BBa_J176171 with an eGFP that we ligated to. The final promoter construction includes the expression vector BBa_J176171, FadBA, and eGFP. Make it to the point: Ex. pFadBA was also obtained from 2013 iGEM Distribution Kit. The promoter was fused with eGFP from pEGFP-N1 and cloned into BBa_J176171 backbone.
The two constructs allow us to investigate the interaction between FadR protein and FadBA promoter in Eukaryotic cells.

FABP1

FABP1, as a promoter, which originally exists in human liver cell, was extracted from human genomic DNA, the gDNA were extracted from HepG2 cells, then via PCR we cloned the promoter sequence. Then, we ligated it to pEGFP-N1 plasmid by replacing the original promoter pCMV. However, as there are two illegal restriction sites, EcoR1 and Pst1, we conducted mutagenesis to remove them for biobrick submission. The construction includes FABP1 promoter, eGFP both placed in BBa_J176171.

GRP78

GRP78 promoter was obtained from a commercial plasmid (Invivogene,pDRIVE_hGRP78). We did PCR to get the promoter and then ligated it to pEGFP-N1 plasmid by replacing the original promoter pCMV. The promoter contains one illegal restriction site, Xba1, therefore we conduced mutagenesis to remove it for biobrick submission. The final construction includes GRP78 promoter inside pEGFP-N1.
Although GRP78 is not only promoted by Fatty Acids, it also can be induced by any other Endoplasmic Reticulum Stress factor.
After transfecting to mammalian cells, these promoters will be induced by Fatty Acids or its oxidation products, leading to expression of eGFP. By comparing the fluorescnece intensity of eGFP using Fluorescent microscopy, we will be able to quantify their expression and determine the desired sensing mechanism, which is most efficient for Glyoxylate genes expression.

Characterization

Every promoter works under different Fatty Acid concentrations, therefore we expose the final constructions to different concentration of Fatty Acid, 50, 150, 250, 350 450 and 550 mM then observed after 3 hrs and 5 hrs after exposure to Fatty Acid. Applying fluorescent microscopy we observed GFP expression and chose the desired promoter.