2019 iGEM

 These are the results of our experiments on the project of creating a hyperstable antibody for intracellular targeting. 

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Gene Design

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We designed the gene as a combination of pET-28a (+) vector and scFv (P5) cell-penetrating peptide. It includes His6-tag so that we can identify it later.

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Cloning

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This was the experiment for April 28th. We have cut and put together the gene of our design and used gel electrophoresis to see if it the process was successful.

Here is an example of the optimal result:

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And...here are our results:

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We can see that not all were successful.

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Transformation & Expression

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Now that we have created the recombinant gene, we transformed it into a competent cell BL21 (DE3), which is exceptional for DNA expression. (Our Experiment on May 26th)

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Here are our colonies grew as a result of our experiment on May 26th.

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Purification

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With our expression finished, we harvested the proteins and used a sonicator to cause cell lysis. Then, we created an elution sample by using Ni-NTA.

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Then in our June 9th experiment, we ran it through affinity chromatography with SDS-PAGE and were able to see a band at 29kDa location, confirming our protein has been successfully made.

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Identification

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In order to make sure, we ran it through western blot and confirmed our protein with the marker His6-tag. This is the June 23rd and July 7th experiment.

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Binding Assay

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Using lysozyme, the target of scFv (P5), we proceeded with the binding assay with sizing exclusion chromatography. This is our July 14th experiment.

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                                                                                                    We were able to see that it                                                                                                        bonded with lysozyme and                                                                                                          done it's purpose.

                                                                                                    Success!

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Cell-penetrating Assay

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Finally, in the experiment we ran twice, first on July 21st and the second on August 11th, in order to really see that our scFv (P5) CPP has penetrated the cell, we did immunostaining.

We used 2 cells, HEK293T and HeLa, and compared it to a control.  

 

 

 

 

 

 

 

 

 

 

 

 

On one cell, we can see the CPP dyed green, and in the merged image, the blue-dyed nucleus (with dapi) is co-localized. So, the CPP-included antibody was able to penetrate the cell.

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08/11 result

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Summary

We were able to successfully:

• Design an antibody for intracellular targeting

• Transform the pET-28a (+) vector with scFv (P5) CPP to BL21 (DE3)

• Confirm the placement of our scFv (P5) CPP after transformation

• Identify our protein with the His6-tag

• See our protein target its objective through binding assay

• Confirm that our protein is able to penetrate cells and function in them

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Unfortunately, we were not successful in:

• Combining the genes (pET-28a (+) and scFv (P5)) ourselves

• Growing the colony as much as we wanted (we could still use it, though)

It was difficult for us to carry out these experiments since it required extensive knowledge; we were confused at times of certain procedures.

Nevertheless, we were able to learn in the process and were proud of the results. Of course, some of the experiments could have gone better: injecting the samples into the agarose gel for gel electrophoresis and western blot was quite challenging, and it took a long time to finish each experiment.