Team:EPF Lausanne/Next steps

From 2013.igem.org

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{{Template:EPFL2013Header}}
{{Template:EPFL2013Header}}
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Up until now we succeded to produce nanoparticles and loading them, to clone a pH sensitive protomer and to engineer a fusion protein between ice nucleation protein (INP) and streptavidin.<br>
+
Up until now, we succeeded to produce nanoparticles and to load them, to clone a pH sensitive promoter and to engineer a fusion protein between ice nucleation protein (INP) and streptavidin.<br>
But we neither had the time to characterize our parts extensively nor to assemble the parts to finalize the Taxi.Coli.<br\>
But we neither had the time to characterize our parts extensively nor to assemble the parts to finalize the Taxi.Coli.<br\>
-
==What we would have done with 1 month more==
+
==What we would have done with 1 more month==
-
 
+
==Nanoparticles==
==Nanoparticles==
-
This part of our project is rather advanced, but there are still some assays that we would have loved to make. For example to test if and how fast the nanoparticles are digested by GelatinaseE and Matrixmetalloprotease 2 (MMP2) we would have performed a fluorescent release assay using a plate reader. First we would have used commercial enzymes, to try if the assay works and maybe adjust the protocol, later with the enzymes produced by our own Taxi.Coli.<br>
+
This part of our project is rather advanced, but there are still some assays that we would have loved to make. For example, to test if and how fast the nanoparticles are digested by GelatinaseE and Matrix metalloprotease 2 (MMP2), we would have performed a fluorescence release assay using a plate reader. First, we would have used commercial enzymes to check that the assay works and maybe adjust the protocol, later with the enzymes produced by the E.coli transformed with the effector plasmid.<br>
-
Eventually we would have also tried to load the nanoparticles with an actual drug.<br>
+
Eventually, we would have also tried to load the nanoparticles with an actual drug.<br>
==Cell surface display of Streptavidin==
==Cell surface display of Streptavidin==
-
We weren't able to proof that the fusion protein between Inp and Streptavidin was exported to the outer membrane. The next step would be to clone a plasmid that encodes a fusion protein between Inp,streptavidin and YFP. Then we would have been able to follow the same immunofluorescent staining protocol we succesfully used to characterized the Biobrick BBa_K523013. We actually started this cloning strategy, but didn't have enough time to optimize the PCRs.
+
We weren't able to prove that the fusion protein between Inp and Streptavidin was exported to the outer membrane. The next step would be to clone a plasmid that encodes a fusion protein between Inp, streptavidin and YFP. Then, we would be able to follow the same immunofluorescent staining protocol we successfully used to characterize the Biobrick BBa_K523013. We actually started this cloning strategy, but didn't have enough time to optimize the PCRs.
-
Additionally we would have inquiered which other proteins could be fused to INP and still be used to bind nanoparticles.<br>
+
<br>
 +
<br>
 +
We actually used the time between the Jamboree in Lyon and the one at MIT, to clone the two plasmids, and did microscopy using an anti YFP antibody. Check out Cell surface display for further information.
 +
The results were promising, but we would need to sequence verify our constructs and repeat the assay several times, improve the antibody concentration and incubation time, for better images.
 +
Additionally, we would have inquired which other proteins could be fused to INP and still be used to bind nanoparticles.<br>
==Sensing/Effector==
==Sensing/Effector==
-
 
+
We successfully cloned a pH sensitive promoter, and proved its functionality, but, with a bit more time, we would have characterized it in more details and would have been able to find its optimal expression conditions. For example, we would have repeated the plate reader experiments with a wider range of pH values and even with different buffers.<br>
-
• Make more plate reader experiments, with more different pHs and differnt Buffers<br>
+
Furthermore, we would have transformed another strain of E.coli (MG1655) from which the promoters (hya and cad) originate with the plasmid. This would assure that the necessary transcription factors and activators are present and can initiate transcription of the superfolded GFP.
-
• Transform other E.coli K12 (MG1655), because the promoter originate from this strain, so maybe the needed xxxy aren't present in the DHalpha strain we use at the moment <br>
+
In parallel, we would have restarted the cloning of the hya and cad promoters with slight variations of sequences, to check for a better inducible system.<br><br>
-
• Start new cloning, with slight variations of the sequence <br>
+
The effector part wasn't very successful, but we would repeat the purification assay of Gelatinase E and MMP2 to make sure that our negative results aren't due to manipulations error. <br>
-
• Redo the purification of the Gelatinase E and of Matrixmetalloprotease2 (MMP2)
+
Additionally, we would have designed new primers to have a fusion protein between the enzymes and GFP which would facilitate to prove its production.
-
• Repeat the Cloning strategy with new primers, because those promoters added a stop codon directly after the enzyme, and before the GFP, so to repeat the cloning, would make the visualization much more easier.<br>
+
<br><br>
-
• Clone the sensing promoter together with the enzyme to make the plasmid that induces enzyme production upon a signal.
+
Eventually, we would clone the sensing promoter and the effector enzymes together into one plasmid. Thus having a plasmid that produces the nanoparticle degrading enzyme when triggered by a pH change, as in the original idea.
-
 
+
==Taxi.Coli==
==Taxi.Coli==
-
• Cloned one of our Biobricks in a backbone that contains another resistance than chloramphenicol to cotransfect bacteria with the Plasmids responsible for Sensing/Effector and the Plasmid used to connect the Nanoparticles.<br>
+
Once all the subparts worked, we would clone one Biobrick made out of our promoter and our gelatinase gene in one backbone. This backbone would then contain a different antibiotic resistance than our Cell-surface display plasmid so that we could co-transform bacteria with the plasmid responsible for sensing/effector and the plasmid that encodes the Inp-streptavidin fusion protein. Then, we would bind the nanoparticles to those Taxi.Coli bacteria.<br>
-
• <br>
+
The last step to complete the proof of principle we wanted to provide is to characterize extensively this final version of Taxi.Coli smart drug delivery.
-
•<br>
+
{{Template:EPFL2013Footer}}
{{Template:EPFL2013Footer}}

Latest revision as of 23:57, 28 October 2013

Taxi.Coli: Smart Drug Delivery iGEM EPFL

Header

Up until now, we succeeded to produce nanoparticles and to load them, to clone a pH sensitive promoter and to engineer a fusion protein between ice nucleation protein (INP) and streptavidin.
But we neither had the time to characterize our parts extensively nor to assemble the parts to finalize the Taxi.Coli.

Contents

What we would have done with 1 more month

Nanoparticles

This part of our project is rather advanced, but there are still some assays that we would have loved to make. For example, to test if and how fast the nanoparticles are digested by GelatinaseE and Matrix metalloprotease 2 (MMP2), we would have performed a fluorescence release assay using a plate reader. First, we would have used commercial enzymes to check that the assay works and maybe adjust the protocol, later with the enzymes produced by the E.coli transformed with the effector plasmid.
Eventually, we would have also tried to load the nanoparticles with an actual drug.

Cell surface display of Streptavidin

We weren't able to prove that the fusion protein between Inp and Streptavidin was exported to the outer membrane. The next step would be to clone a plasmid that encodes a fusion protein between Inp, streptavidin and YFP. Then, we would be able to follow the same immunofluorescent staining protocol we successfully used to characterize the Biobrick BBa_K523013. We actually started this cloning strategy, but didn't have enough time to optimize the PCRs.

We actually used the time between the Jamboree in Lyon and the one at MIT, to clone the two plasmids, and did microscopy using an anti YFP antibody. Check out Cell surface display for further information. The results were promising, but we would need to sequence verify our constructs and repeat the assay several times, improve the antibody concentration and incubation time, for better images. Additionally, we would have inquired which other proteins could be fused to INP and still be used to bind nanoparticles.

Sensing/Effector

We successfully cloned a pH sensitive promoter, and proved its functionality, but, with a bit more time, we would have characterized it in more details and would have been able to find its optimal expression conditions. For example, we would have repeated the plate reader experiments with a wider range of pH values and even with different buffers.
Furthermore, we would have transformed another strain of E.coli (MG1655) from which the promoters (hya and cad) originate with the plasmid. This would assure that the necessary transcription factors and activators are present and can initiate transcription of the superfolded GFP. In parallel, we would have restarted the cloning of the hya and cad promoters with slight variations of sequences, to check for a better inducible system.

The effector part wasn't very successful, but we would repeat the purification assay of Gelatinase E and MMP2 to make sure that our negative results aren't due to manipulations error.
Additionally, we would have designed new primers to have a fusion protein between the enzymes and GFP which would facilitate to prove its production.

Eventually, we would clone the sensing promoter and the effector enzymes together into one plasmid. Thus having a plasmid that produces the nanoparticle degrading enzyme when triggered by a pH change, as in the original idea.

Taxi.Coli

Once all the subparts worked, we would clone one Biobrick made out of our promoter and our gelatinase gene in one backbone. This backbone would then contain a different antibiotic resistance than our Cell-surface display plasmid so that we could co-transform bacteria with the plasmid responsible for sensing/effector and the plasmid that encodes the Inp-streptavidin fusion protein. Then, we would bind the nanoparticles to those Taxi.Coli bacteria.
The last step to complete the proof of principle we wanted to provide is to characterize extensively this final version of Taxi.Coli smart drug delivery.