The plasmid from the ER1 samples was isolated by the [https://2013.igem.org/Team:NTNU-Trondheim/Protocols#Transformed_cells Promega Wizard Plus SV Minipreps DNA Purification System A1460] and sequenced. Figure below shows the alignment of the sequencing results with the reference DNA sequence.
The plasmid from the ER1 samples was isolated by the [https://2013.igem.org/Team:NTNU-Trondheim/Protocols#Transformed_cells Promega Wizard Plus SV Minipreps DNA Purification System A1460] and sequenced. Figure below shows the alignment of the sequencing results with the reference DNA sequence.
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center>[[file:ER1.jpg|400px]]</center>
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<center>[[file:ER1.jpg|400px]]</center>
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<center>figure: Aligment of tat_GFP_l_RFP (ER1) with reference DNA. </center>
<center>figure: Aligment of tat_GFP_l_RFP (ER1) with reference DNA. </center>
PCR for amplification of tat, plasmid backbone, GFP and RFP
There were essentially four DNA pieces that we wanted to combine together to make a construct: The tat signal sequence followed by GFP, a small linker region and RFP put into a plasmid backbone. As our cloning techniques rely on overlapping DNA fragments we used mostly primers with overhengs in the PCR reactions. As templates we used the biobricks <partinfo>BBa_E1010</partinfo> for RFP, <partinfo>BBa_E0040</partinfo> for GFP, <partinfo>BBa_J01101</partinfo> for the plasmid backbone and genomic DNA from ‘’Escherichia coli’’ strain ER2566 for the tat signal sequence.
Table: Primers applied in creating the tat_GFP_l_RFP construct. Lowercase letters indicate DNA that anneal to the template whereas the uppercase letters indicate DNA that serves as an overhang.
The linker region is going to be only 36 bp long and will therefore be created by overlapping overhengs on the reverse primer of GFP and forward primer of RFP. The primers for the plasmid backbone is designed to include the TetR repressible promoter (<partinfo>BBa_R0040</partinfo>), RBS (<partinfo>BBa_B0034</partinfo>) and two terminators (<partinfo>BBa_B0010</partinfo> and <partinfo>BBa_B0012</partinfo>) in the PCR product.
All of the PCR products were treated with the enzyme DpnI that digests methylated DNA and purified by the QIAquick PCR Purification kit.
Gibson Assembly and transformation
Our overlapping DNA fragments; tat, GFP, RFP and plasmid backbone was cloned together by Gibson Assembly and transformed into ‘’E.coli’’ strain ER2566 cells. The photograph below shows two of the resulting colonies (named ER1 and ER2) from this transformation.
figure: Two of the transformed colonies with the tat_GFP_l_RFP construct. Hereby named ER1 and ER2.
figure: Aligment of tat_GFP_l_RFP (ER1) with reference DNA.
The sequence align almost perfectly. There seems to be some sort of extra insert at the linker region, but this insert is dividable by 3, so the reading frame is maintained. This is supported by the fact that the colonies with this construct is red (see figure above).
Red ER1-cells in liquid media was immobilized in agar and then viewed in a confocal microscope for seeing if RFP was localized in the periplasm. The results can be seen in the two figures below:
There is no indication that the red fluorescence is more concentrated in the periplasm, as we should expect due to the transport through the tat transport pathway.
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