Difference between revisions of "Part:BBa K1761001:Experience"

 
(Experience TU Eindhoven 2015)
 
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This experience page is provided so that any user may enter their experience using this part.<BR>Please enter
 
This experience page is provided so that any user may enter their experience using this part.<BR>Please enter
 
how you used this part and how it worked out.
 
how you used this part and how it worked out.
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===Applications of BBa_K1761001===
 
===Applications of BBa_K1761001===
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== Application TU Eindhoven 2015 ==
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Implementing the amber stop codon TAG for building in the an azide-functionalized amino acid.  The protein is used together with part BBa_K1761002 [https://parts.igem.org/Part:BBa_K1761002] in a pETDuet-1 vector. This vector, together with the pEVOL-pAzF (coding for tRNA + tRNA synthethase) were inserted in BL21(DE3) competent cells by  double transformation.
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The proteins were expressed and the DBCO-PEG4-TAMRA, Fluorescence and bioluminescence confirmation were successful (see the main page and BBa_K1761002 [https://parts.igem.org/Part:BBa_K1761002]).
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Additionally, two different oligos with functionalized DBCO groups were clicked on membrane proteins. Fluorescently labeled complementary strands were incubated with the bacteria as an extra confirmation of the protein expression and click reaction (see Figure 1).
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[[File:TU_Eindhoven_Oligos_verification.png]]
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''Figure 1: To verify whether the click reaction has occured with DBCO-modified DNA, we incubate the cells with DBCO-functionalized complementary strands. If the DBCO-modified DNA clicks, the fluorescently labeled DBCO-functionalized complementary strands will anneal with the clicked strands. In that case, the cells will remain fluorescent after a few washing steps.''
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By using strand displacement (see Figure 2), the two membrane proteins are brought in close proximity (see Figure 3).
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[[File:TU_Eindhoven_DNA_strand_displacement.png]]
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''Figure 2: Schematic overview of DNA strand displacement. DNA Strand displacement is initiated when the input binds to the toehold region of a partially hybridized DNA strand. After the initiation, branch migration takes place and the input fully hybridizes with its perfect match, yielding the output.''
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[[File:TU_Eindhoven_DNA_strand_displacement_System.png]]
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''Figure 3: The membrane proteins can be brought in close proximity by clicking oligonucleotides on the loops and adding a long strand complementary to both oligonucleotides. It is expected that this results in a measurable signal. The longer strand is functionalized with a toehold region. Upon addition of a strand perfectly complementary to the longer strand, the system disassembles and the signal will fade out.''
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===User Reviews===
 
===User Reviews===

Latest revision as of 18:02, 16 September 2015

This experience page is provided so that any user may enter their experience using this part.
Please enter how you used this part and how it worked out.


Applications of BBa_K1761001

Application TU Eindhoven 2015

Implementing the amber stop codon TAG for building in the an azide-functionalized amino acid. The protein is used together with part BBa_K1761002 [1] in a pETDuet-1 vector. This vector, together with the pEVOL-pAzF (coding for tRNA + tRNA synthethase) were inserted in BL21(DE3) competent cells by double transformation. The proteins were expressed and the DBCO-PEG4-TAMRA, Fluorescence and bioluminescence confirmation were successful (see the main page and BBa_K1761002 [2]).

Additionally, two different oligos with functionalized DBCO groups were clicked on membrane proteins. Fluorescently labeled complementary strands were incubated with the bacteria as an extra confirmation of the protein expression and click reaction (see Figure 1).

TU Eindhoven Oligos verification.png

Figure 1: To verify whether the click reaction has occured with DBCO-modified DNA, we incubate the cells with DBCO-functionalized complementary strands. If the DBCO-modified DNA clicks, the fluorescently labeled DBCO-functionalized complementary strands will anneal with the clicked strands. In that case, the cells will remain fluorescent after a few washing steps.

By using strand displacement (see Figure 2), the two membrane proteins are brought in close proximity (see Figure 3).

TU Eindhoven DNA strand displacement.png

Figure 2: Schematic overview of DNA strand displacement. DNA Strand displacement is initiated when the input binds to the toehold region of a partially hybridized DNA strand. After the initiation, branch migration takes place and the input fully hybridizes with its perfect match, yielding the output.

TU Eindhoven DNA strand displacement System.png

Figure 3: The membrane proteins can be brought in close proximity by clicking oligonucleotides on the loops and adding a long strand complementary to both oligonucleotides. It is expected that this results in a measurable signal. The longer strand is functionalized with a toehold region. Upon addition of a strand perfectly complementary to the longer strand, the system disassembles and the signal will fade out.


User Reviews

UNIQb8ccf360f679df51-partinfo-00000000-QINU UNIQb8ccf360f679df51-partinfo-00000001-QINU