Difference between revisions of "Part:BBa K2243029"

Latest revision as of 15:41, 1 November 2017

Int2 attB_855F_int2 attP

To test the influence of attBP sites of Int2 to terminator ECK120010855 (abbreviation: 855) in the forward 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 1: / Line 1: @@
-__NOTOC__
 <partinfo>BBa_K2243029 short</partinfo>
 To test the influence of attBP sites of Int2 to terminator ECK120010855 (abbreviation: 855) in the forward direction.
-<!-- Add more about the biology of this part here
-===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:
+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|Fig.1 Terminator Strength Induced with 0.1mM 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 IPTG 0.1M.png|200px|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 IPTG 1M.png|200px|thumb|center|Terminator Strength Induced with 1M 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]]
-. We then characterized the inversion efficiency.
+<!-- -->
+<h2>Terminator Reference Table</h2>
-We transformed the testing system and expression system of corresponding recombinase into one single cell to see if the inversions happened, and if the leakage expression of recombinases would impact the system. In the end, couples of terminators and recombinases with near complete inversions and minimal leakage were selected.
+[[Media:Peking_trt.xlsx]]
-Microplate spectrophotometer was used to conduct preliminary measurements. Bacterial culture was added into 96-well plate(200ul for each well). OD600 and fluorescence intensity were measured. Background OD600 and fluorescence of plate, culture medium ,and autofluorescence should be eliminated through setting control groups. Fluorescence intensity/OD600 was cauculated using the net fluorescence intensity and net OD600. The transcription barrier strength of attBP/Terminator can be characterized quantitatively referring to the Ts formula.