Difference between revisions of "Part:BBa K2243030"

Revision as of 15:29, 1 November 2017

Int2 attB_855R_int2 attP

To test the influence of attBP sites of Int2 to terminator ECK120010855 (abbreviation: 855) in the reverse direction.

Usage

We constructed this part to characterize the recombination efficiency of the integrase Int2. It consists of the terminator ECK120030221 (abbreviation: 221) in the reverse direction flanked by attB and attP sites of recombinase Int2. Upon recombination, the orientation of the terminator changes. As a result, expression of downstream sequence is blocked.

Biology

The attP site of Int2 is used to integrate phage DNA at the host attB site, 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 terminator, attB and attP sites by oligo synthesis.

Characterization

We first characterized the terminator strength using the following formula:

Ts=〖GFP〗of the random sequence/〖[GFP]〗with the terminator

Fig.1 Terminator Strength Induced with 0.1mM IPTG

Fig.2 Terminator Strength Induced with 1mM IPTG

And we sifted out 6 desirable unidirectional terminators without potential cryptic promotor in both orientations.

Fig.3 Terminator Strength Induced with 0.1M IPTG

Fig.4 Forward and Reverse Terminator Strength Ratio Induced with 0.1mM and 1mM IPTG

Terminator Reference Table

Media:Peking_trt.xlsx

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
COMPATIBLE WITH RFC[12]
21
COMPATIBLE WITH RFC[21]
23
COMPATIBLE WITH RFC[23]
25
COMPATIBLE WITH RFC[25]
1000
INCOMPATIBLE WITH RFC[1000]
Illegal SapI site found at 22

@@ Line 4: / Line 4: @@
 To test the influence of attBP sites of Int2 to terminator ECK120010855 (abbreviation: 855) in the reverse direction.
-===Usage and Biology===
 <!-- -->
-Usage
+<h2>Usage</h2>
 We constructed this part to characterize the recombination efficiency of the integrase Int2. It consists of the terminator ECK120030221 (abbreviation: 221) in the reverse direction flanked by attB and attP sites of recombinase Int2. Upon recombination, the orientation of the terminator changes. As a result, expression of downstream sequence is blocked.
 <!-- -->
-Biology
+<h2>Biology</h2>
 The attP site of Int2 is used to integrate phage DNA at the host attB site, 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 terminator, attB and attP sites by oligo synthesis.
 <!-- -->
-===Characterization===
+<h2>Characterization</h2>
 We first characterized the terminator strength using the following formula:
@@ Line 26: / Line 23: @@
 Ts=〖GFP〗of the random sequence/〖[GFP]〗with the terminator
-[[File:Peking_TS_0.1mM.png|800px|thumb|center|Terminator Strength Induced with 0.1mM IPTG]]
+[[File:Peking_TS_0.1mM.png|800px|thumb|center|Fig.1 Terminator Strength Induced with 0.1mM IPTG]]
-[[File:Peking_TS_1mM.png|800px|thumb|center|Terminator Strength Induced with 1mM IPTG]]
+[[File:Peking_TS_1mM.png|800px|thumb|center|Fig.2 Terminator Strength Induced with 1mM IPTG]]
 And we sifted out 6 desirable unidirectional terminators without potential cryptic promotor in both orientations.
-[[File:Peking ratio.png|600px|thumb|center|Terminator Strength Induced with 0.1M IPTG]]
+[[File:Peking ratio.png|600px|thumb|center|Fig.3 Terminator Strength Induced with 0.1M IPTG]]
-[[File:Peking TS good terminators.png|600px|thumb|center|Forward and Reverse Terminator Strength Ratio Induced with 0.1mM and 1mM IPTG]]
+[[File:Peking TS good terminators.png|600px|thumb|center|Fig.4 Forward and Reverse Terminator Strength Ratio Induced with 0.1mM and 1mM IPTG]]
 <!-- -->
-Terminator Reference Table
+<h2>Terminator Reference Table</h2>
 [[Media:Peking_trt.xlsx]]