PCR for sequence isolation.

PCR 25 ul Mix for Mammalian expression Biobricks

-6.75 µl – Molecular grade water
-1 µl – DNA template (1 ng)
-1.25 µl – Primer F (100 uM)
-1.25 µl – Primer R (100 uM)
-2.25 µl – DMSO 99%
-12.5 µl – NEB Q5 High Fidelity 2X Master Mix

PCR programs

NeoR

F1Ori

CMV

BGHPA

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Mammalian Cells Transfection

Add 50µl of OPTI-MEM (GIBCO) and 12 µl of Lipofectamine2000 Invitrogen reagent to a microtube. (Solution 1). Incubate at room temperature for 5 minutes.

Add 50µl of Optimen and 40µl (100ng) of plasmid to a microtube (Solution 2). Incubate at room temperature for 5 minutes.

Mix Solutions 1 and 2. Incubate at room temperature for 5 minutes →(Solution 3).

Cell preparation.

1) Wash MARC145 Cells with OPTI-MEM. Repeat two times.
2) Add 1.8ml of OPTI-MEM to the cells.
3) Mix solution 3 with MARC145 cells solution and incubate 4 hours with 6% CO2.
4) Change medium for 2ml fresh OPTI-MEM medium to eliminate Lipofectamine 2000 Invitrogen.
5) Change to 5ml of complete DMEM-10% neonatal serum and add 12.5 µl Geniticin (100µg/ml).

7-dehydratase insertion in pcDNA 3.1 Myc-His A

7-dehydratase was inserted in pcDNA 3.1 Myc-His A. Since our enzyme sequence design has the iGEM preffix and suffix, the only restriction available to insert it into the plasmid was with NotI. This means that the further ligation can be sense or anti-sense. The correct ligation was corroborated with a restriction and was then transfected in monkey kidney cells.

pcDNA 3.1 Myc-His A is an expression plasmid that contains BGHPA, a constitutive promoter and an origin of replication that work in mammalian cells. To see if the plasmid was working correctly, a cassette was expressed with GFP to see if the plasmid worked in a specific eukaryotic cell line (Marc145), so the enzyme could be inserted and the analysis of functionality of the enzyme by substrate degradation could be started.

Image 1.Cells before (left) and after (right) transfection with plasmid pcDNA 3.1 Myc-His A with 7-dehydratase gene using lipofectamine as a transfecting agent.

As fluorescence of this enzyme cannot be detected, its characterization is going to be determined by an antibiotic resistance at a certain concentration (Neomycin), where cells are able to keep dividing, except for the ones without the resistance gene.

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Recombinant Protein Expression

Each protein was inserted in Escherichia coli through pPROEX B which is a bacterial expression plasmid. This plasmid was used due to its characteristics, which include TRC promoter inducible by IPTG and has a 6x histidine tag in N-terminal end.

In the case of cholesterol oxidase, the enzyme was successfully introduced in the plasmid mentioned before. The protein wasn’t overexpressed by induction with IPTG, so no further analysis was made because this enzyme is well characterized and there is more information about it in BRENDA Enzymes as EC 1.1.3.6 – cholesterol oxidase, Chromobacterium sp.

Oxoacyl reductase was analyzed by SDS-PAGE in a 15% acrylamide gel. First, main cultures of the different colonies grown in the plate were inoculated in 40 ml of LB with ampicillin (100 ug/ml). After a few hours in the shaker, optical density was measured repeatedly until it was between 0.5 and 0.65, considering this measurement our time zero. Right after it, IPTG 1 mM was added to start the overexpression of the protein. Each hour, the absorbance of each culture was registered until the time six.

Table 1. Different absorbances measured each hour of the cultures of oxoacyl reductase.

The samples taken each hour were centrifuged 5 min/13500 rpm to concentrate the pellet in the bottom of the microtube. The supernatant was thrown into a waste glass, and the pellets were resuspended in different volumes of Laemmli buffer, depending on the value of absorbance obtained. The criteria used to determine the volume of buffer, was taking into consideration the absorbance of the time zero, which would represent the 100 percent of buffer added (100 ul in this sample), and going up or down depending of the absorbances of the other samples. The different volumes are now shown.

Table 2. Volumes of Laemmli buffer depending of the absorbance value of each sample.

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NeoR characterization

The scope of our project is to express a new synthetic pathway in mammalian cells in order to make them able to metabolize 7-ketocholesterol. Just like bacteria, mammalian cells also have a selective gene to identify successfully transformed organisms. NeoR is a gene that encodes an aminoglycoside 3'-phosphotransferase enzyme, which provides a resistance to Neomycin and its derivatives. The antibiotic that will be used to select the successfully transformed mammalian cells is G418®, a Neomycin derivative which only affects mammalian cells.

However, this exceeded the possibilities given the time constrains. Therefore a characterization in a mammalian cell culture was not done due to time limitations. The NeoR gene was characterized using an E.coli culture and Neomycin as selective antibiotic. The NeoR gene was obtained by PCR from pcDNA3.1(-)/ Myc-His A, and following iGEM instructions, the gene was introduced in the plasmid pSB1C3 as it is shown in the following picture.

Procedure

The characterization was made using two groups: the samples and a control group. The samples were made using an E.coli DH5-α inoculum, transformed with the NeoR gene inserted in psB1C3 using the constitutive promoter BBa_K823012. The control group used an untransformed E.coli DH5-α inoculum.

Both groups consisted on thirteen essay tubes with 5 ml of LB media each one with a different concentration on Neomycin as shown in table 1 and 2. The tubes were cultured on a shaker for 18 hours at 250 rpm/37ºC. Afterwards, each tube had its optical density measured at 600 nm using as LB media as a blank with the same antibiotic concentration. Five neomycin concentrations were chosen to perform petri dish cultures but only with the trouble group to perform a C.F.U. count.

Table 1. Control group without neomycin

Table 2. Positive Group with neomycin

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