Difference between revisions of "Part:BBa K2243028"
 
(5 intermediate revisions by the same user not shown)
Line 1: Line 1:
  
−
__NOTOC__
+
 
 
<partinfo>BBa_K2243028 short</partinfo>
 
<partinfo>BBa_K2243028 short</partinfo>
  
 
To test the influence of attBP sites of Int2 to terminator ECK120030221 (abbreviation: 221) in the reverse direction.
 
To test the influence of attBP sites of Int2 to terminator ECK120030221 (abbreviation: 221) in the reverse direction.
  
−
<!-- Add more about the biology of this part here
+
 
 
===Usage and Biology===
 
===Usage and Biology===
 +
 +
<!-- -->
 +
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
 +
 +
[[File:Peking_TS_0.1mM.png|800px|thumb|center|Terminator Strength Induced with 0.1mM IPTG]]
 +
 +
[[File:Peking_TS_1mM.png|800px|thumb|center|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 TS good terminators.png|600px|thumb|center|Forward and Reverse Terminator Strength Ratio Induced with 0.1mM and 1mM IPTG]]
 +
 +
 +
 +
<!-- -->
 +
Terminator Reference Table
 +
 +
[[Media:Peking_trt.xlsx]]
 +
 +
 +
 +
  
 
<!-- -->
 
<!-- -->

Latest revision as of 15:41, 1 November 2017


Int2 attB_221R_int2 attP

To test the influence of attBP sites of Int2 to terminator ECK120030221 (abbreviation: 221) in the reverse direction.


Usage and Biology

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

Terminator Strength Induced with 0.1mM IPTG
Terminator Strength Induced with 1mM IPTG

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

Terminator Strength Induced with 0.1M IPTG
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