Team:ITESM-CEM/Project/Experiments

From 2014.igem.org

(Difference between revisions)
Line 113: Line 113:
However the scope of our project exceeded the possibilities given the time constrains. Therefore a characterization in a 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 pcDNA3.1myc his A, and following iGEM instructions, the gene was introduced in the plasmid psB1C3 as it is shown in the following picture.<br><br></p>
However the scope of our project exceeded the possibilities given the time constrains. Therefore a characterization in a 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 pcDNA3.1myc his A, and following iGEM instructions, the gene was introduced in the plasmid psB1C3 as it is shown in the following picture.<br><br></p>
-
<img src="https://static.igem.org/mediawiki/2014/e/ef/Plasmido_BBa_K1313004.jpg" width="400" height="369" hspace="20">
+
<img src="https://static.igem.org/mediawiki/2014/e/ef/Plasmido_BBa_K1313004.jpg" width="450" height="369" hspace="20">
<h4>Procedure</h4>
<h4>Procedure</h4>
       <p tyle="text-align: justify; text-justify: inter-word;">The characterization was made using two groups: the trouble group and the control group. The trouble group was 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. <br><br>
       <p tyle="text-align: justify; text-justify: inter-word;">The characterization was made using two groups: the trouble group and the control group. The trouble group was 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. <br><br>

Revision as of 06:15, 17 October 2014

TEC-CEM | Project

ITESM-CEM | Enzy7-K me

Project 3014

 

The Experiments

If you choose to create a model during your project, please write about it here. Modeling is not an essential part of iGEM, but we encourage any and all teams to model some aspect of their project. See previous "Best Model" awards for more information.

Experiment One

Lorem ipsum dolor sit amet, consectetur adipiscing elit. Vivamus fringilla porta nisi sed dictum. Aliquam at rutrum nisl. Ut eros quam, condimentum sed pretium et, euismod vitae tellus. Curabitur eu blandit massa. Vestibulum at euismod purus. Sed quis pretium ligula. Cras non tristique nulla. Nunc finibus risus non purus malesuada viverra. Cras bibendum augue eu lorem faucibus, nec auctor mi iaculis. Vivamus sagittis, mi non gravida accumsan, odio dui pellentesque leo, a varius dui mauris eu diam. Maecenas laoreet tellus sed porta efficitur.

Back to top ↑

Experiment Two

Etiam tempus mi pulvinar purus iaculis bibendum. Vivamus vel risus eu enim volutpat finibus. Nullam bibendum est sit amet arcu lobortis, id laoreet ex vestibulum. Curabitur fringilla eleifend lacus, nec ornare nibh imperdiet sed. Maecenas vel velit consectetur, tempus libero quis, consectetur ex. Nulla porttitor pharetra velit. Curabitur tristique, dolor ut sodales euismod, diam diam ultricies arcu, at tincidunt tellus neque in felis. Etiam ut tempor ligula. Sed at dui sapien.

Back to top ↑

Experiment Three

Proin aliquam nibh id elementum pellentesque. Suspendisse mollis est ut felis sagittis mollis. Lorem ipsum dolor sit amet, consectetur adipiscing elit. Etiam accumsan ex ante, quis lobortis erat fermentum ac. Sed et egestas libero. Donec id diam vitae leo consequat interdum. Ut in sem in quam pretium finibus vitae non lectus.

Back to top ↑

Experiment Four

Curabitur sed erat ante. Maecenas viverra odio sit amet ultrices facilisis. Nullam lobortis mi quam, sit amet placerat erat eleifend eget. Mauris commodo sem tortor, quis blandit orci laoreet vel. In hac habitasse platea dictumst. Curabitur molestie eu dolor ac bibendum. Duis fermentum tellus et dui dapibus, sed feugiat dolor sollicitudin. Nulla malesuada, tortor quis blandit dictum, odio risus rutrum sapien, varius pharetra dui tellus sed lectus. Sed eget magna at justo tempus hendrerit. Nulla in odio nec libero maximus finibus.

Back to top ↑

NeoR characterization

The scope of our Project is to express in mammalian cells the new synthetic pathway able to metabolize 7-ketocholesterol. Just like bacteria, mammalian cells also need a selective gene to identify successfully transformed organisms. NeoR is a gene that encodes an aminoglycoside 3'-phosphotransferase enzyme, which provides in theory 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 the scope of our project exceeded the possibilities given the time constrains. Therefore a characterization in a 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 pcDNA3.1myc 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 trouble group and the control group. The trouble group was 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. Afterwards each tube had its optical density measured at 600 nm using as blank LB media at 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.

Back to top ↑