Contents

C1 (<a href="http://parts.igem.org/Part:BBa_K1366101 ">BBa_K1366101</a>)

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This construct contains the sequence homologies of 50 bp to delete the lpp gene (Braun’s lipoprotein). It includes an ampicillin resistance gene and the sequences for the LuxR synthesis (when this protein is coupled with the homoserine lactone (AHL), it activates the transcription of genes controlled by the lux box). The flippase recognition target (FRT) sequences included are used to remove the antibiotic resistance with a specific recombinase. For more detail on the role of AHL and FRT, refer to <a href="module3.html"> Module 3 </a>.

Its general structure is as follows:

<figure> <a href="ITESM14_BiobrickC1.png" data-lightbox="I1" data-title="Figure B1: Structure construct 1"><img class="img img-responsive" style="margin:0px auto;display:block width:20%;" src="ITESM14_BiobrickC1.png"></a>

<figcaption> Figure B1: Structure construct 1</Figcaption>

</figure> </section>

C2 (<a href="http://parts.igem.org/Part:BBa_K1366102 ">BBa_K1366102</a>)

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This construct contains the sequence homologies to delete the msbB gen (LPS myristic acid moiety carrier), a kanamycin resistance gene and the sequences for the Lux I production (AHL synthase).

Its general structure is as follows:

<figure> <a href="ITESM14_BiobrickC2.png" data-lightbox="I2" data-title="Figure B2: Structure of construct 3"><img class="img img-responsive"style="margin:0px auto;display:block " src="ITESM14_BiobrickC2.png"></a>

<figcaption> Figure B2: Structure of construct 3</Figcaption>

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p42 (<a href="http://parts.igem.org/Part:BBa_K1366109 ">BBa_K1366109</a>)

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This part allows the production of a recombinant pIII protein linked with a pep42 peptide. This peptide change the tropism of the virus M13 and enables it to bind to a receptor called GRP78 that it is highly present in cancer cells. The binding between pep42 and GRP78 receptor allows the bacteriophage to penetrate the cancer cells and this initiates the transfection of DNA mediated with bacteriophages.

The biobrick contains the CDS inside pSB1C3 backbone.

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C4 (GFP)(<a href="http://parts.igem.org/Part:BBa_K1366106 ">BBa_K1366106</a>)

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This construct will be used as a proof of concept to demonstrate that the phage is capable of transfecting DNA to cancerous cells, and, at the same time, that these cells are able to express the phagemid.

Its general structure is as follows:

<figure> <a href="ITESM14_HTERT-GFP_Map.png" data-lightbox="I2" data-title="Figure B3: Structure of construct 4"><img class="img img-responsive"style="margin:0px auto;display:block " src="ITESM14_HTERT-GFP_Map.png"></a>

<figcaption> Figure B2: Structure of construct 3</Figcaption>

</figure> </section>

C5 (Apoptin)(<a href="http://parts.igem.org/Part:BBa_K1366104 ">BBa_K1366104</a>)

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The 13kDa protein Apoptin gets phosphorilated in treonin 108 only in cancerous cells, which enables it to be translocated to the nucleous and subsequently trigger apoptosis. This effector will be expressed under a CMV constitutive promoter because of the high selectivity of the protein to cancer cells.

<figure> <a href="ITESM14_CMV-apoptina_Map.png" data-lightbox="I2" data-title="Figure B4: Structure of construct 3"><img class="img img-responsive"style="margin:0px auto;display:block " src="ITESM14_CMV-apoptina_Map.png"></a>

<figcaption> Figure B4: Structure of construct 3</Figcaption>

</figure> </section>

Survivin siRNA

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siRNA wil be used to decrease survivin expression, so cell arrest and apoptosis of cancerous cells are expected. Two siRNAs found in literature and another one custom designed are used to observe the difference between the effectors. These constructs are expressed with hTERT promoter, which is only activated in cancerous cell lines that have telomerase overexpression.

C6 (siRNA1)(<a href=" http://parts.igem.org/Part:BBa_K1366105">BBa_K1366105</a>)

<figure> <a href="ITESM14_SiRNA1_Map.png" data-lightbox="siRNA1" data-title="Fig. B5: Structure of construct 6."><img class="img img-responsive" style="margin:0px auto;display:block width:20%;" src="ITESM14_SiRNA1_Map.png"></a>

<figcaption> Fig. B5: Structure of construct 6. </Figcaption>

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C7 (siRNA2)(<a href=" http://parts.igem.org/Part:BBa_K1366106">BBa_K1366106</a>)

<figure> <a href="ITESM14_SiRNA2_Map.png" data-lightbox="siRNA2" data-title="Fig. B6: Structure of construct 7."><img class="img img-responsive" style="margin:0px auto;display:block width:20%;" src="ITESM14_SiRNA2_Map.png"></a>

<figcaption> Fig. B6: Structure of construct 7. </Figcaption>

</figure>

C8 (siRNA3)(<a href=" http://parts.igem.org/Part:BBa_K1366107">BBa_K1366107</a>)

<figure> <a href="ITESM14_SiRNA3_Map.png" data-lightbox="siRNA3" data-title="Fig. B7: Structure of construct 8."><img class="img img-responsive" style="margin:0px auto;display:block width:20%;" src="ITESM14_SiRNA3_Map.png"></a>

<figcaption> Fig. B7: Structure of construct 8. </Figcaption>

</figure> </section>

C9 (Apoptin - siRNA)(<a href=" http://parts.igem.org/Part:BBa_K1366108">BBa_K1366108</a>)

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At last, both effectors were included in the same construct, for us to observe if there is synergy, antagonism, or have no effect against each other.

<figure> <a href="ITESM14_Apoptin-siRNA_Map.png" data-lightbox="Apoptin - siRNA" data-title="Fig. B8: Structure of construct 9."><img class="img img-responsive" style="margin:0px auto;display:block width:20%;" src="ITESM14_Apoptin-siRNA_Map.png"></a>

<figcaption> Fig. B8: Structure of construct 9. </Figcaption>

</figure> </section> </section>