Difference between revisions of "Part:BBa K1962000"

Revision as of 20:38, 16 October 2016

Colicin Ia

This sequence codes for the full length Colicin Ia bacteriocin (anti-bacterial toxin). Colicin Ia is a protein that is used by species of E. coli in order to kill closely related species of E. coli in certain circumstances e.g. competition. The producer cell normally contains a protein which confers immunity to this toxin, which in this case is (BBa_K1962001). And this protein is believed to form a complex with the cytotoxic domain of the bacterocin rendering it inactive. When this bacteriocin is secreted the immunity protein dissociates from the complex leaving the cytotoxic domain active resulting in the pore forming activity of this bacteriocin returning and having effect on the target cells. The pore forming activity of this bacteriocin results in the depolarisation of the bacterial inner membrane resulting in the collapse of the protonmotive force.

Usage and Biology

Colicin Ia is synthesised on the ribosome and translocation occurs by the TonB system as this is a group B colicin. Expression of these proteins is tightly regulated by the REcA and LexA proteins involved in the SOS response. The expression of colicins can be inhibited by the binding of LexA to the SOS promoter and this action is reversed by the binding of RecA to LexA. This colicin uses two Cir receptors in order to enter the cell. One for binding and another for translocation. The TonB protein forms a complex with the other cell membrane proteins, Exb8 and ExbD and this complex provides the energy required for the translocation of the cytotoxic domain into the cell.

Below is the structure of colicin Ia, in green is the receptor binding domain, in red the translocation domain and in blue the cytotoxic domain. This was cloned into pSB1C3 with the Biobrick prefix and suffix.

Cloning Strategy

Our aim was to create a device which would express Colicin Ia in response to pH and bile salts. We cloned the Ia-Immunity protein downstream of a pH sensitive promoter P_asr (BBa_K1231000) and a bile salt sensitive promoter P_acrRA (BBa_K1231001) to generate the composite parts (BBa_K1962015) and (BBa_K1962011), respectively. Next we wanted to clone Colicin Ia downstream of the Ia-Immunity protein however after several attempts we were unable to clone colicin Ia into both of these devices. This may have been due to the fact that this colicin exerted too much of a toxic effect in E. coli and therefore preventing us from obtaining any positive transformants. In order to overcome this problem we decided to clone Colicin Ia downstream of the pBAD promoter, this allowed us to repress expression with 0.5% D-glucose during the cloning steps. However, the only transformants we obtained contained a TAA (stop codon) at position BLA and therefore we would not have expression of the full Colicin Ia.

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
INCOMPATIBLE WITH RFC[25]
Illegal NgoMIV site found at 132
Illegal AgeI site found at 1777
1000
COMPATIBLE WITH RFC[1000]

@@ Line 3: / Line 3: @@
 <partinfo>BBa_K1962000 short</partinfo>
-This sequence codes for the full length Colicin Ia bacteriocin (anti-bacterial toxin). Colicin Ia is a protein that is used by species of <i>E. coli</i> in order to kill closely related species of <i>E. coli</i> in certain circumstances e.g. competition. The producer cell normally contains a protein which confers immunity to this toxin, which in this case is (BBa_K1962001). And this protein is believed to form a complex with the cytotoxic domain of the bacterocin rendering it inactive. When this bacteriocin is secreted the immunity protein dissociates from the complex leaving the cytotoxic domain active resulting in the pore forming activity of this bacteriocin returning and having effect on the target cells. The pore forming activity of this bacteriocin results in the depolarisation of the bacterial inner membrane resulting in the collapse of the protonmotive force.
+This sequence codes for the full length Colicin Ia bacteriocin (anti-bacterial toxin). Colicin Ia is a protein that is used by species of <i>E. coli</i> in order to kill closely related species of <i>E. coli</i> in certain circumstances e.g. competition. The producer cell normally contains a protein which confers immunity to this toxin, which in this case is (<partinfo>BBa_K1962001</partinfo>). And this protein is believed to form a complex with the cytotoxic domain of the bacterocin rendering it inactive. When this bacteriocin is secreted the immunity protein dissociates from the complex leaving the cytotoxic domain active resulting in the pore forming activity of this bacteriocin returning and having effect on the target cells. The pore forming activity of this bacteriocin results in the depolarisation of the bacterial inner membrane resulting in the collapse of the protonmotive force.
 <b>Usage and Biology</b>
@@ Line 13: / Line 13: @@
 <b>Cloning Strategy </b>
-Our aim was to create a device which would express colicin Ia in response to pH and bile salts. We cloned the Ia-Immunity protein downstream of a pH sensitive promoter Pasr (BBa_) and a bile salt sensitive promoter PacrRA (BBa) to generate the composite part (BBa) and (BBa), respectively. Next we wanted to clone Colicin Ia downstream of the Ia-Immunity protein however after several attempts we were unable to clone colicin Ia into both of these devices. This may have been due to the fact that this colicin exerted too much of a toxic effect in E. coli and therefore preventing us from obtaining any positive transformants. In order to overcome this problem we decided to clone Colicin Ia downstream of the pBAD promoter, this allowed us to repress expression with 0.5% D-glucose during the cloning steps. However, the only transformants we obtained contained a TAA (stop codon) at position BLA and therefore we did not have expression of our full Colicin Ia.
+Our aim was to create a device which would express Colicin Ia in response to pH and bile salts. We cloned the Ia-Immunity protein downstream of a pH sensitive promoter P<sub>asr</sub> (<partinfo>BBa_K1231000</partinfo>) and a bile salt sensitive promoter P<sub>acrRA</sub> (<partinfo>BBa_K1231001</partinfo>) to generate the composite parts (<partinfo>BBa_K1962015</partinfo>) and (<partinfo>BBa_K1962011</partinfo>), respectively. Next we wanted to clone Colicin Ia downstream of the Ia-Immunity protein however after several attempts we were unable to clone colicin Ia into both of these devices. This may have been due to the fact that this colicin exerted too much of a toxic effect in <i>E. coli</i> and therefore preventing us from obtaining any positive transformants. In order to overcome this problem we decided to clone Colicin Ia downstream of the pBAD promoter, this allowed us to repress expression with 0.5% D-glucose during the cloning steps. However, the only transformants we obtained contained a TAA (stop codon) at position BLA and therefore we would not have expression of the full Colicin Ia.
-, 2015