Difference between revisions of "Part:BBa K3137014"

Latest revision as of 20:26, 21 October 2019

responce circuit

Usage and Biology

The responce gene circuit is composed of an inducible promoter PputA(BBa_K3137002) and anti-phage protein genes of the incubation period (about 5 min), and an inducible promoter PglcF (BBa_K3137003) and a toxic protein gene protegrin-1(BBa_K62800) of the burst period (about 20 min).

When E. coli is infected by a phage, responce gene circuit is induced to express. When a phage infects E. coli into the incubation period (about 5 min), the inducible promoter PputA will induce the bacteria to express the resistant proteins (BBa_K3137006)(BBa_K3137005).

If the resistant proteins successfully resist phage infection, the inducible promoter PglcF will not induce the expression of downstream gene. If the resistant proteins fail to kill phages, it means that phages will continue to infect bacteria, when it comes to the burst period (around 20 min) of phage infection, the inducible promoter PglcF will activate the expression of downstream toxic protein gene so that the bacteria will lyse cells before the assembly of phages.

We performed the phage infection assay on the LB agar plate, and got very good resistance.

Figure 1. Resistance for T4 phage of E. coli BL21-pET28a-Pputa-abpAB-gntR-PglcF-P-1.

In addition, we inoculated E. coli BL21 and recombinant E. coli BL21-pET28a-PputA-abpAB-gntR-PglcF-P-1 in LB liquid medium to raise the logarithmic growth phase, i.e. OD 0.6-0.8 . Then the fresh phage solution was inoculated at the same time and culture was continued for 1-2 h. As a result, it was found that the recombinant grew well.

Figure 2. Resistance test for T4 phage of E. coli BL21-pET28a-Pputa-abpAB-gntR-PglcF-P-1 in flasks.

Sequence and Features

Assembly Compatibility:

10
INCOMPATIBLE WITH RFC[10]
Illegal EcoRI site found at 1280
Illegal EcoRI site found at 2048
Illegal PstI site found at 1313
Illegal PstI site found at 2705
Illegal PstI site found at 3785
12
INCOMPATIBLE WITH RFC[12]
Illegal EcoRI site found at 1280
Illegal EcoRI site found at 2048
Illegal PstI site found at 1313
Illegal PstI site found at 2705
Illegal PstI site found at 3785
Illegal NotI site found at 4987
21
INCOMPATIBLE WITH RFC[21]
Illegal EcoRI site found at 1280
Illegal EcoRI site found at 2048
Illegal BglII site found at 1619
Illegal BglII site found at 2679
Illegal BamHI site found at 481
23
INCOMPATIBLE WITH RFC[23]
Illegal EcoRI site found at 1280
Illegal EcoRI site found at 2048
Illegal PstI site found at 1313
Illegal PstI site found at 2705
Illegal PstI site found at 3785
25
INCOMPATIBLE WITH RFC[25]
Illegal EcoRI site found at 1280
Illegal EcoRI site found at 2048
Illegal PstI site found at 1313
Illegal PstI site found at 2705
Illegal PstI site found at 3785
Illegal NgoMIV site found at 4391
1000
COMPATIBLE WITH RFC[1000]

@@ Line 3: / Line 3: @@
 <partinfo>BBa_K3137014 short</partinfo>
-[[Image:Respond circuit.png|400px]]
+[[Image:Respond circuit2.png|400px]]
-The respond gene circuit is composed of an inducible promoter PputA and an anti-phage protein gene of the incubation period (about 5 min), and an inducible promoter PglcF and a toxic protein gene kilP of the outbreak period (about 20 min).
+===Usage and Biology===
-When E. coli is infected by a phage, both report and respond gene circuits are induced to express at the same time. When a phage infects E. coli into the incubation period (about 5 min), the inducible promoter PputA will induce the bacteria to express the resistant protein and the green fluorescent protein gene gfp.
+The responce gene circuit is composed of an inducible promoter PputA([https://parts.igem.org/Part:BBa_K3137002# BBa_K3137002]) and anti-phage protein genes of the incubation period (about 5 min), and an inducible promoter PglcF ([https://parts.igem.org/Part:BBa_K3137003# BBa_K3137003]) and a toxic protein gene protegrin-1([https://parts.igem.org/Part:BBa_K62800# BBa_K62800]) of the burst period (about 20 min).
-If the resistant protein successfully resists phage infection, the inducible promoter PglcF will not induce the expression of downstream gene. If the resistant protein fails to kill phages, it means that phages will continue to infect bacteria, when it comes to the outbreak period (around 20 min) of phage infection, the inducible promoter PglcF will activate the red fluorescent protein gene mCherry and the expression of downstream toxic protein gene so that the bacteria will display red fluoresce and lyse cells before the assembly of phages.
+When <i>E. coli</i> is infected by a phage, responce gene circuit is induced to express. When a phage infects <i>E. coli</i> into the incubation period (about 5 min), the inducible promoter PputA will induce the bacteria to express the resistant proteins ([https://parts.igem.org/Part:BBa_K3137006# BBa_K3137006])([https://parts.igem.org/Part:BBa_K3137005# BBa_K3137005]).
+If the resistant proteins successfully resist phage infection, the inducible promoter PglcF will not induce the expression of downstream gene. If the resistant proteins fail to kill phages, it means that phages will continue to infect bacteria, when it comes to the burst period (around 20 min) of phage infection, the inducible promoter PglcF will activate the expression of downstream toxic protein gene so that the bacteria will lyse cells before the assembly of phages.
+We performed the phage infection assay on the LB agar plate, and got very good resistance.
+[[Image:respond1.png|500px|center|thumb|Figure 1. Resistance for T4 phage of <i>E. coli</i> BL21-pET28a-Pputa-<i>abpAB</i>-<i>gntR</i>-PglcF-<i>P-1</i>.]]
+In addition, we inoculated E. coli BL21 and recombinant E. coli BL21-pET28a-PputA-abpAB-gntR-PglcF-P-1 in LB liquid medium to raise the logarithmic growth phase, i.e. OD 0.6-0.8 . Then the fresh phage solution was inoculated at the same time and culture was continued for 1-2 h. As a result, it was found that the recombinant grew well.
+[[Image:respond2.png|500px|center|thumb|Figure 2. Resistance test for T4 phage of <i>E. coli</i> BL21-pET28a-Pputa-<i>abpAB</i>-<i>gntR</i>-PglcF-<i>P-1</i> in flasks.]]