Difference between revisions of "Part:BBa K2332003"

Latest revision as of 15:01, 25 October 2017

tRNA-Pyl (pylT)

This composite part encodes tRNA-Pyl (pyIT), a tRNA with the anticodon CUA able to incorporate the photocaged amino acid pyrrolysine (Ne-methyl-L-lysine), into proteins in E. coli. Pyrolysyl-tRNA synthetase first loads the tRNA with pyrrolysine present in the media. The charged pyIT can then incorporate pyrrolysine into proteins where the corresponding sense codon TAG, is present in the mRNA during translation.

In our project, this construct enables the photocaging and inactivation of our SpyCatcher variants (Intimin'-SpyCatcher and GFP-SpyCatcher) with the introduction of Ne-methyl-L-lysine instead of the reactive lysine required for its binding to SpyTag. SpyTag (13 amino acids) and SpyCatcher (138 amino acids, 15 kDa) are protein binding partners that originate from CnaB2 (immunoglobulin-llike collagen adhesin domain) of the FbaB protein, found in the invasive strains of S. pyogenes. For post-translational light control, amberless E. coli cells must express this construct as well as pyrrolysyl-tRNA synthetase and the UAA, Ne-methyl-L-lysine, must be supplemented in the media. The pyrrolysyl-tRNA synthetase catalyses the acylation of the suppressor tRNACUA with the UAA. During translation, the UAG amber codon in the mRNA is recognised by the acylated tRNACUA and the UAA will be added to the growing polypeptide chain. Post-translational light control allows a precise spatio-temporal control of protein function.

Figure 1: Agarose gel electrophoresis of pSB1C3-pylT NotI digestions. EcoRI-PstI (EP) digested pylT (308bp) was ligated to EP digested and DpnI treated pSB1C3. The ligation product was transformed onto E. coli cells, cultures were made of resulting colonies. DNA from cultures was isolated (miniprepped) and NotI digested. 2-log DNA ladder (lanes 1 and 8), NotI digested pSB1C3-Pyl colonies (lanes 2-6), NotI digested pSB1C3-mRFP (lane 7). Only the colony in lane 6 contains the pSB1C3-Pyl (tRNA-Pyl, 308bp) construct (shown with the red arrow).

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
INCOMPATIBLE WITH RFC[12]
Illegal NheI site found at 7
Illegal NheI site found at 30
21
COMPATIBLE WITH RFC[21]
23
COMPATIBLE WITH RFC[23]
25
COMPATIBLE WITH RFC[25]
1000
COMPATIBLE WITH RFC[1000]

@@ Line 6: / Line 6: @@
 Pyrolysyl-tRNA synthetase first loads the tRNA with pyrrolysine present in the media. The charged pyIT can then incorporate pyrrolysine into proteins where the corresponding sense codon TAG, is present in the mRNA during translation.
-In our project, this construct enables the photocageing and inactivation of our SpyCatcher variants ([https://parts.igem.org/Part:BBa_K2332025 Intimin'-SpyCatcher] and [https://parts.igem.org/Part:BBa_K2332042 GFP-SpyCatcher]) with the introduction of Ne-methyl-L-lysine instead of the reactive lysine required for its binding to SpyTag. SpyTag (13 amino acids) and SpyCatcher (138 amino acids, 15 kDa) are protein binding partners that originate from CnaB2 (immunoglobulin-llike collagen adhesin domain) of the FbaB protein, found in the invasive strains of S. pyogenes. For post-translational light control, amberless E. coli cells must express this construct as well as [https://parts.igem.org/Part:BBa_K1223013 pyrrolysyl-tRNA synthetase] and the UAA, Ne-methyl-L-lysine, must be supplemented in the media. The pyrrolysyl-tRNA synthetase catalyses the acylation of the suppressor tRNACUA with the UAA. During translation, the UAG amber codon in the mRNA is recognised by the acylated tRNACUA and the UAA will be added to the growing polypeptide chain. Post-translational light control allows a precise spatio-temporal control of protein function.
+In our project, this construct enables the photocaging and inactivation of our SpyCatcher variants ([https://parts.igem.org/Part:BBa_K2332025 Intimin'-SpyCatcher] and [https://parts.igem.org/Part:BBa_K2332042 GFP-SpyCatcher]) with the introduction of Ne-methyl-L-lysine instead of the reactive lysine required for its binding to SpyTag. SpyTag (13 amino acids) and SpyCatcher (138 amino acids, 15 kDa) are protein binding partners that originate from CnaB2 (immunoglobulin-llike collagen adhesin domain) of the FbaB protein, found in the invasive strains of S. pyogenes. For post-translational light control, amberless E. coli cells must express this construct as well as [https://parts.igem.org/Part:BBa_K1223013 pyrrolysyl-tRNA synthetase] and the UAA, Ne-methyl-L-lysine, must be supplemented in the media. The pyrrolysyl-tRNA synthetase catalyses the acylation of the suppressor tRNACUA with the UAA. During translation, the UAG amber codon in the mRNA is recognised by the acylated tRNACUA and the UAA will be added to the growing polypeptide chain. Post-translational light control allows a precise spatio-temporal control of protein function.
-[[File:tRNA-Pyl-gel.png|thumb|center|500px| Figure 1: Agarose gel electrophoresis of pSB1C3-pylT NotI digestions. EcoRI-PstI (EP) digested pylT (308bp) was ligated to EP digested and DpnI treated pSB1C3. The ligation product was transformed onto E. coli cells, cultures were made of resulting colonies. DNA from cultures was isolated (miniprepped) and NotI digested. 2-log DNA ladder (lanes 1 and 8), NotI digested pSB1C3-Pyl colonies (lanes 2-6), NotI digested pSB1C3-mRFP (lane 7). Only the colony in lane 6 contains the pSB1C3-Pyl (tRNA-Pyl, 308bp) construct.]]
+[[File:tRNA-Pyl-gel.png|thumb|center|500px| Figure 1: Agarose gel electrophoresis of pSB1C3-pylT NotI digestions. EcoRI-PstI (EP) digested pylT (308bp) was ligated to EP digested and DpnI treated pSB1C3. The ligation product was transformed onto E. coli cells, cultures were made of resulting colonies. DNA from cultures was isolated (miniprepped) and NotI digested. 2-log DNA ladder (lanes 1 and 8), NotI digested pSB1C3-Pyl colonies (lanes 2-6), NotI digested pSB1C3-mRFP (lane 7). Only the colony in lane 6 contains the pSB1C3-Pyl (tRNA-Pyl, 308bp) construct (shown with the red arrow).]]