Difference between revisions of "Part:BBa K2243006:Design"
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− | === | + | ===Usage=== |
− | + | We constructed this part to characterize the recombination efficiency of the recombinase Bxb1 gp35 (BBa_K2243012). It consists of a constitutive promoter (BBa_J23119) flanked by attB and attP sites of recombinase Bxb1 gp35. Different orientation of attB and attP allows the sequence to be flipped, excised, or inserted between recognition sites, which makes it useful for gene editing. Upon recombination, the orientation of the constitutive promoter change. As a result, expression of downstream sequence is shut down, and upstream sequence is transcribed. | |
+ | ===Biology=== | ||
+ | J23119 is the consensus sequence of a combinatorial constitutive promoter library family. The attP site of Mycobacteriophage Bxb1 is used to integrate phage DNA at the host attB site of Mycobacterium smegmatis, generating the recombinant junctions attL and attR. DNA cleavage and re-ligation occur at the central crossover region at attB and attP, which allows the sequence to be flipped, excised, or inserted between recognition sites. We obtained the promoter, attB and attP sites by oligo synthesis. | ||
+ | |||
+ | ===Design note=== | ||
+ | We construct this structure by Gibson Assembly. | ||
+ | |||
+ | ===Characterize=== | ||
+ | We used this part to characterize the recombination efficiency of the integrase Bxb1 gp35 (BBa_K2243012). | ||
+ | <html> | ||
+ | <body> | ||
+ | |||
+ | <img src="https://static.igem.org/mediawiki/2017/f/fc/Peking_flipflop_fig_5.svg" /> | ||
+ | |||
+ | </body> | ||
+ | </html> | ||
+ | |||
+ | Figure 1. The flipping efficiency of Bxb1 gp35 with different RBS. | ||
+ | |||
+ | Figure 2. The flipping efficiency of Bxb1 gp35 with different concentration of inducer. | ||
===Source=== | ===Source=== | ||
− | Mycobacterium | + | Mycobacterium Phage Bxb1 |
===References=== | ===References=== |
Latest revision as of 14:55, 29 October 2017
Bxb1 attB_J23119_Bxb1 attP
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 102
Illegal NheI site found at 125 - 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 41
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 61
Illegal BsaI.rc site found at 160
Usage
We constructed this part to characterize the recombination efficiency of the recombinase Bxb1 gp35 (BBa_K2243012). It consists of a constitutive promoter (BBa_J23119) flanked by attB and attP sites of recombinase Bxb1 gp35. Different orientation of attB and attP allows the sequence to be flipped, excised, or inserted between recognition sites, which makes it useful for gene editing. Upon recombination, the orientation of the constitutive promoter change. As a result, expression of downstream sequence is shut down, and upstream sequence is transcribed.
Biology
J23119 is the consensus sequence of a combinatorial constitutive promoter library family. The attP site of Mycobacteriophage Bxb1 is used to integrate phage DNA at the host attB site of Mycobacterium smegmatis, generating the recombinant junctions attL and attR. DNA cleavage and re-ligation occur at the central crossover region at attB and attP, which allows the sequence to be flipped, excised, or inserted between recognition sites. We obtained the promoter, attB and attP sites by oligo synthesis.
Design note
We construct this structure by Gibson Assembly.
Characterize
We used this part to characterize the recombination efficiency of the integrase Bxb1 gp35 (BBa_K2243012).
Figure 1. The flipping efficiency of Bxb1 gp35 with different RBS.
Figure 2. The flipping efficiency of Bxb1 gp35 with different concentration of inducer.
Source
Mycobacterium Phage Bxb1