Difference between revisions of "Part:BBa K1897016"

Latest revision as of 21:34, 18 October 2016

LuxR + GFP

This part is made by 3A ligation (see: https://parts.igem.org/Help:Assembly/3A_Assembly) of LuxR BBa_K1897008 and green fluorescent protein (GFP) BBa_K1897014. LuxR is derived from Vibrio fischeri while GFP is derived from Aequeora victoria.

Usage and Biology

This part is intended to be used with the external addition of N-(3-oxo-hexanoyl)-homoserine lactone (AHL) in order to result in the expression of GFP.

The LuxR produced will bind to pLuxR promoter BBa_R0062 only in the presence of sufficient amounts of AHL. Normally in Vibrio fischeri, LuxI produces the AHL required. In this case however, since no LuxI is present in this part, external AHL has to be added in order to activate transcription of GFP. The GFP produced can then be detected when excited at 396 nm or 475 nm, resulting in fluroescence at 503 or 509 nm (Mishin et al., 2010).

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
INCOMPATIBLE WITH RFC[12]
Illegal NheI site found at 949
Illegal NheI site found at 972
21
COMPATIBLE WITH RFC[21]
23
COMPATIBLE WITH RFC[23]
25
INCOMPATIBLE WITH RFC[25]
Illegal AgeI site found at 84
Illegal AgeI site found at 992
1000
INCOMPATIBLE WITH RFC[1000]
Illegal BsaI.rc site found at 740

Construction of LuxR+GFP

LuxR+GFP was made via 3A ligation of LuxR BBa_K1897008 and GFP BBa_K1897014. This was done by cutting GFP (BBa_K1897014) with EcoRI (at the Biobrick prefix) and SpeI (at the Biobrick suffix) and cutting LuxR (BBa_K1897008) with XbaI (at the Biobrick prefix) and PstI (at the Biobrick suffix). At the same time, the pSB1C3 backbone was cut with EcoRI and PstI. The 3 fragments were ligated together using T4 ligase, with a scar site formed between GFP and LuxR due to the compatible overhangs created by SpeI and XbaI. This resulted in an end product of a plasmid with GFP and LuxR joined together.

Verification of LuxR+GFP

LuxR+GFP was verified via sequencing results, and the sequencing of the insert showed that it was of the correct sequence.

Besides the verification of the LuxR+GFP part, we also verified the part by adding external AHL to the bacteria harboring the plasmid. With no AHL added, fluorescence was very faint, and when 100 μM AHL was added, the fluorescence increased (Figure 1, Figure 2).

Figure 1: Induction of GFP production with/without AHL. Left Column: Green fluorescence microscopy images of LuxR+GFP bacteria of control with 0 μM AHL added (top) and with 100 μM AHL added (bottom). Right Column: Bright field fluorescence microscopy images of LuxR+GFP bacteria of control with 0 μM AHL added (top) and with 100 μM AHL added (bottom).

GFP levels were also measured using a microplate reader in the presence or absence of AHL, and the results obtained over a period of 6 hours can be seen in Figure 2. The level of fluorescence is higher for induced (100 μM AHL) than non induced (0 μM AHL), indicating that the addition of AHL induces expression of GFP from pLuxR.

Figure 2: GFP quantifications via microplate reader of LuxR+GFP for 6 hours is shown in the presence of 0 or 100 μM AHL incubation. The level of fluorescence is higher for induced (100 μM AHL) than non induced (0 μM AHL), indicating that the addition of AHL induces expression of GFP from pLuxR.

@@ Line 3: / Line 3: @@
 <partinfo>BBa_K1897016 short</partinfo>
-This part is made by 3A ligation of LuxR <partinfo>BBa_K1897008</partinfo> and GFP <partinfo>BBa_K1897014</partinfo>.
+This part is made by 3A ligation (see: https://parts.igem.org/Help:Assembly/3A_Assembly) of LuxR <partinfo>BBa_K1897008</partinfo> and green fluorescent protein (GFP) <partinfo>BBa_K1897014</partinfo>. LuxR is derived from <i>Vibrio fischeri</i> while GFP is derived from <i>Aequeora victoria</i>.
-<!-- Add more about the biology of this part here
 ===Usage and Biology===
+This part is intended to be used with the external addition of N-(3-oxo-hexanoyl)-homoserine lactone (AHL) in order to result in the expression of GFP.
+The LuxR produced will bind to pLuxR promoter <partinfo>BBa_R0062</partinfo> only in the presence of sufficient amounts of AHL. Normally in <i>Vibrio fischeri</i>, LuxI produces the AHL required. In this case however, since no LuxI is present in this part, external AHL has to be added in order to activate transcription of GFP. The GFP produced can then be detected when excited at 396 nm or 475 nm, resulting in fluroescence at 503 or 509 nm (Mishin <i>et al.</i>, 2010).
 <!-- -->
 <span class='h3bb'>Sequence and Features</span>
 <partinfo>BBa_K1897016 SequenceAndFeatures</partinfo>
+===Construction of LuxR+GFP===
+LuxR+GFP was made via 3A ligation of LuxR <partinfo>BBa_K1897008</partinfo> and GFP <partinfo>BBa_K1897014</partinfo>. This was done by cutting GFP (<partinfo>BBa_K1897014</partinfo>) with EcoRI (at the Biobrick prefix) and SpeI (at the Biobrick suffix) and cutting LuxR (<partinfo>BBa_K1897008</partinfo>) with XbaI (at the Biobrick prefix) and PstI (at the Biobrick suffix). At the same time, the pSB1C3 backbone was cut with EcoRI and PstI. The 3 fragments were ligated together using T4 ligase, with a scar site formed between GFP and LuxR due to the compatible overhangs created by SpeI and XbaI. This resulted in an end product of a plasmid with GFP and LuxR joined together.
+===Verification of LuxR+GFP===
+LuxR+GFP was verified via sequencing results, and the sequencing of the insert showed that it was of the correct sequence.
+Besides the verification of the LuxR+GFP part, we also verified the part by adding external AHL to the bacteria harboring the plasmid. With no AHL added, fluorescence was very faint, and when 100 μM AHL was added, the fluorescence increased (Figure 1, Figure 2).
+[[File:BBa_K1897016_LuxR+GFP_v2.png|200px|thumb|centre|<b>Figure 1:</b> Induction of GFP production with/without AHL. Left Column: Green fluorescence microscopy images of LuxR+GFP bacteria of control with 0 μM AHL added (top) and with 100 μM AHL added (bottom). Right Column: Bright field fluorescence microscopy images of LuxR+GFP bacteria of control with 0 μM AHL added (top) and with 100 μM AHL added (bottom).]]
+GFP levels were also measured using a microplate reader in the presence or absence of AHL, and the results obtained over a period of 6 hours can be seen in Figure 2. The level of fluorescence is higher for induced (100 μM AHL) than non induced (0 μM AHL), indicating that the addition of AHL induces expression of GFP from pLuxR.
+[[File:BBa_K1897016_LuxR+GFP_graph.png|200px|thumb|centre|<b>Figure 2:</b> GFP quantifications via microplate reader of LuxR+GFP for 6 hours is shown in the presence of 0 or 100 μM AHL incubation. The level of fluorescence is higher for induced (100 μM AHL) than non induced (0 μM AHL), indicating that the addition of AHL induces expression of GFP from pLuxR.]]