Difference between revisions of "Part:BBa K5121027:Design"
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Although in vivo, the reb genes are transcriptionally independent, here they are encoded in a polycistronic fashion. To make it sortase A-compatible, this part contains an added LPETG motif at the C terminus of rebA. This modification was made using a two-fragment PCR of the original reb1 plasmid (BBa_K5121011) using primers with complementary overhangs to add in 5’-ctcccagaaacagga-3’ between the stop codon and the rest of the rebA open reading frame (basic part: BBa_K5121019) (Figure 3). Rather than amplifying the whole plasmid in one reaction, the plasmid was amplified in two sections — each containing one of the two complementary primers and a primer within the kanamycin resistance gene — followed by Gibson assembly of the two fragments to reconstruct the full plasmid. This avoided the annealing of the complementary primers. The kanamycin resistance gene was chosen as the site of the second set of primers to ensure only cells with correctly assembled fragments at this site were viable — reducing the possible set of incorrectly assembled fragments. | Although in vivo, the reb genes are transcriptionally independent, here they are encoded in a polycistronic fashion. To make it sortase A-compatible, this part contains an added LPETG motif at the C terminus of rebA. This modification was made using a two-fragment PCR of the original reb1 plasmid (BBa_K5121011) using primers with complementary overhangs to add in 5’-ctcccagaaacagga-3’ between the stop codon and the rest of the rebA open reading frame (basic part: BBa_K5121019) (Figure 3). Rather than amplifying the whole plasmid in one reaction, the plasmid was amplified in two sections — each containing one of the two complementary primers and a primer within the kanamycin resistance gene — followed by Gibson assembly of the two fragments to reconstruct the full plasmid. This avoided the annealing of the complementary primers. The kanamycin resistance gene was chosen as the site of the second set of primers to ensure only cells with correctly assembled fragments at this site were viable — reducing the possible set of incorrectly assembled fragments. | ||
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| + | <img src="https://static.igem.wiki/teams/5121/rebleptg/reba/fig3sortaseusyd.png" width="80%"> | ||
| + | <figcaption><b>Figure 3. Plasmid map of the Reb1 plasmid.</b> Primer targeting sites that were used to add in the rebA C-terminal LPETG motif are shown as annotations and zoomed-in alignments. Sortase A can be used for the conjugation of compounds — such as drugs — onto this part.</figcaption> | ||
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===Source=== | ===Source=== | ||
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===References=== | ===References=== | ||
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Revision as of 08:32, 2 October 2024
Reb1: RebA C-terminal LPETG
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 803
Illegal NheI site found at 865 - 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 745
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
Design Notes
Although in vivo, the reb genes are transcriptionally independent, here they are encoded in a polycistronic fashion. To make it sortase A-compatible, this part contains an added LPETG motif at the C terminus of rebA. This modification was made using a two-fragment PCR of the original reb1 plasmid (BBa_K5121011) using primers with complementary overhangs to add in 5’-ctcccagaaacagga-3’ between the stop codon and the rest of the rebA open reading frame (basic part: BBa_K5121019) (Figure 3). Rather than amplifying the whole plasmid in one reaction, the plasmid was amplified in two sections — each containing one of the two complementary primers and a primer within the kanamycin resistance gene — followed by Gibson assembly of the two fragments to reconstruct the full plasmid. This avoided the annealing of the complementary primers. The kanamycin resistance gene was chosen as the site of the second set of primers to ensure only cells with correctly assembled fragments at this site were viable — reducing the possible set of incorrectly assembled fragments.
