Team:Hong Kong HKUST/Project/module3

Introduction

In our project ,we will introduce bacterial enzymes to mammalian cell to modify metabolic pathway. However, unlike bacteria, citric acid cycle in mammalian cells is compartmentalized in mitochondria. The ACE proteins should be targeted to mitochondria for their functionality. To do so, we fused ACE enzymes with Mitochondrial Leader Sequence (MLS).

Mechanism of Mitochondrial Leader Sequence

MLS will be attached to the N-terminal of enzyme, and bind to the receptor protein on mitochondrial membrane, and diffuse to contact site where inner membrane & outer membrane fuse, then bring the ACE enzyme into mitochondria. Afterward, it will be cleaved , leaving the enzyme in mitochondria.

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.