PCR's for gene isolation
Gel 1.High Fidelity PCRs Electrophoresis Gel . Well content: 2) NeoR PCR(786pb) 3)CMV Promoter PCR(588bp) 4) BGHPA PCR (228bp).
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Biobricks in plasmid psB1C3
BGHPA
The ligation (view Material and Methods) of BGHPA was made with J04450 (psB1C3 with RFP protein). The BB_J04450 by itself produces red colonies and grows in the antibiotic Cloramphenicol. After the ligation and transformation, only the white colonies were selected. An extraction from a white colony growing on 50ml LB Cam+ was made in order to perform gel electrophoresis as shown in figure B.
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Figure A. Isolated BGHPA transformed colony.
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Figure B. BGHPA in plasmid psB1C3 gel electrophoresis. Lane 2.
CMV
The ligation (view material and methods) of CMV was made with J04450 (psB1C3 with RFP protein). The BB_J04450 by itself produces red colonies and grows in the antibiotic Cloramphenicol. After the ligation and transformation, only the white colonies were selected. An extraction from a white colony growing on 50ml LB Cam+ was made in order to perform gel electrophoresis as shown in figure B.
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Figure A. Isolated CMV transformed colony.
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Figure B. CMV in plasmid psB1C3 gel electrophoresis. Lanes 1-3.
NeoR
The ligation (view material and methods) of NeoR was made with J04450 (psB1C3 with RFP protein). The BB_J04450 by itself produces red colonies and grows in the antibiotic Cloramphenicol. After the ligation and transformation 8 (Figure A), only the white colonies were selected. An extraction from a white colony growing on 50ml LB Cam+ was made in order to perform gel electrophoresis as shown in Figure B. On lane 8, NeoR extraction is shown with the three bands isoforms, they are barely visible because the plasmid extraction was diluted 5 fold.
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Figure A. Isolated NeoR transformed colony.
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Figure B. NeoR in plasmid psB1C3 gel electrophoresis.
Lanes:
2-4. NeoR in psB1C3 digestion with XhoI.
6-8. NeoR in psB1C3.
10-12. CMV in psB1C3.
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Gene isolation testing via digestion
CMV
A digestion proof of the CMV construction in pSB1C3 is shown on lane 1 (Figure A); when compared to the analysis performed in silico (Figure B), both band patterns coincided.
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Figure A. Lane 5. CMV digestion with XhoI.
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Figure B. In silico CMV-psB1C3 digestion.
NeoR
On the second, third and fourth lanes we can see the NeoR in pSB1C3 digestion with XhoI (Figure A). Even though dim bands can be seen because of the dilution of the original extraction, the banding pattern complies with what was expected from the in silico digestion of this construct (Figure B). The longest DNA fragment is undigested plasmid.
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Figure A. Lanes 2-4. NeoR digestion with XhoI.
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Figure B. In silico NeoR-psB1C3 digestion.
BGHPA
A digestion proof of the BGHPA construction in pSB1C3 is shown on lane 5 (Figure A). When compared to the analysis performed in silico (Figure B), both band patterns coincided.
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Figure A. Lane 5. BGHPA digestion with XhoI.
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Figure B. In silico BGHPA-psB1C3 digestion.
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Mammalian Cells Transfection
Once a construction which included the biobricks CMV promoter, and BGHPA polyadenylation signal was built, using GFP (BBa_E0240) as a marker of gene expression; the plasmid was transfected into monkey kidney cells (MARC-145 cell line) using the protocol previously described (see Materials and Methods) in order to assess its functionality in an eukaryotic environment. A photograph of the cells before transfection, taken with an inverted phase microscope, is shown in figure 1.
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Figure 1. MARC-145 cells before transfection as seen with an inverted phase microscope.
Another photograph was taken using the inverted phase microscope 48 hours after transfection. Two control experiments were run: one without lipofectamine, and another one with lipofectamine and no plasmid. Figure 2 shows a comparison between both controls and the experiment. Cell growth exists for all samples because Geneticin selection cannot yet be measured.
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Figure 2. From left to right, control cells treated with no lipofectamine, control cells treated with lipofectamine and no plasmid and cells exposed to lipofectamine and plasmid. Photographs were taken using an inverted phase microscope. Cell growth is seen for all the experiments.
Since cell growth exists for all cultures, the experiment was treated with UV light at 395nm in order to asses for fluorescence, which would be a proof of gene expression. The results of this analysis are shown in figure 3, where fluorescence can be seen. However, it can be easily seen that transfection efficiency is not as high as predicted.
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Figure 3. Lipofected cells containing the GFP-expressing device exposed to UV radiation. Lipofection efficiency is low but construction funtionality is proven by the fluorescence observed.
Because gene expression exists, as seen in figure 3, we can correctly infer that both biobricks: CMV promoter, and BGHPA polyadenylation signal are properly working. If this was not the case, transcription would either not be possible or be unable to cease, because of a lack of termination signals. The existence of fluorescence reveals the presence of a functional GFP protein, which in turn proves that both bioparts are functional.
Finally, figure 4 shows the results for transfection of a construction including CMV promoter, BGHPA, and one of the enzymes cloned by the team (7-dehidratase). However, due to a lack of time, characterization of gene expression for this construction was not performed.
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Figure 4. Lipofected cells containing the 7-dehydratase expressing plasmid construction as seen after transfection.
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Recombinant Protein Expression
The samples were loaded in a 15% acrylamide gel, using Precision Plus Protein TM Dual Color Standards, for 20 minutes/90 V for the stacking gel and 60 minutes/150V for the resolving gel. The results are now presented:
Only the samples shown in the image before were the ones that presented notable bands that represent our protein of interest. As expected, the most remarked band is the one of the time 3, which means that inductions was taken correctly and more protein was produced, in other words, the protein was overexpressing. The band marked with the arrow represents a protein that weights approximately 34 kDa, which corresponds to the molecular weight of oxoacyl reductase according to ExPASy’s Compute pI/MW tool.
7-dehydratase was analyzed by SDS-PAGE in a 15% acrylamide gel using Precision Plus Protein TM Unstained Standards, for 20 minutes/90 V for the stacking gel and 90 minutes/110V for the resolving gel. Four samples were taken, including one before and after induction with IPTG, one from the soluble phase and one from the inclusion bodies; all prepared with Laemmli buffer. The results are shown in the image below.
No analysis of solubility was realized due to the quantity of protein. It was supposed to be done exactly the same than oxoacyl reductase, as the protein was found in a notable way in the inclusion bodies as shown in the lane 5.
For both enzymes no further work was done. After the identification of each of them, and after the analysis of solubility, the proteins have to be purified by affinity chromatography with a Invitrogen Ni-NTA Agarose column, taking the advantage of the histidine tag added to the protein. After the purification, enzymatic parameters would be determined by the interaction of the enzymes with the substrate; 7β-Hydroxycholesterol for cholesterol oxidase, and 5-Cholesten-3β-ol-7-one for 7-dehydratase and oxoacyl reductase.
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NeoR Results
Results obtained from experimentation.
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Enzymes
BB_K1313000
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Figure 1. Growth of isolated white colony showing a successful transformation with cholesterol oxidase enzyme in LB plate with chloramphenicol (35 mg/ul).
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Figure 2.Gel electrophoresis 0.8% agarose. Extraction of cholesterol oxidase enzyme ligated in psB1C3 (September 5th, 2014) Lane M: Marker Invitrogen DNA Ladder 1kb Plus (5 uL), Lane 5: Cholesterol oxidase in pSB1C3 plasmid. (5uL)
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Figure 3. In silico restriction analysis of psB1C3 with cholesterol oxidase. Fragments generated with PstI and EcoRI.
Comparing the analysis of restriction in silico of cholesterol oxidase in pSB1C3 (Figure 3) against the electrophoresis gel shown in the image below, the two expected bands can be seen (2033 bp, 956 bp. Shown in boxes.) This shows that the enzyme was correctly inserted into the plasmid.
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Image 4.Gel electrophoresis 0.8% agarose. Cholesterol oxidase restriction with EcoRI and PstI (September 23th, 2014) Lane M: Marker Invitrogen DNA Ladder 1kb Plus (5 uL), Lane 1: Undigested cholesterol oxidase (5 uL), Lane 2 and 3: empty, Lane 4: Cholesterol oxidase enzyme digestion with EcoRI and PstI (10 uL) .
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