Difference between revisions of "Part:BBa K1897016"
Line 3: Line 3:
 
<partinfo>BBa_K1897016 short</partinfo>
 
<partinfo>BBa_K1897016 short</partinfo>
  
−
This part is made by 3A ligation (see: https://parts.igem.org/Help:Assembly/3A_Assembly) of LuxR <partinfo>BBa_K1897008</partinfo> and GFP <partinfo>BBa_K1897014</partinfo>. LuxR is derived from <i>Vibrio fischeri</i> while GFP is derived from <i>Aequeora victoria</i>.
+
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>.
  
 
===Usage and Biology===
 
===Usage and Biology===

Revision as of 20:34, 15 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 using the restriction enzyme sites SpeI on the biobrick suffix of LuxR (BBa_K1897008) and XbaI on the biobrick prefix of GFP (BBa_K1897014). After cutting those two restriction enzyme site, ligation was performed using T4 ligase and a scar site is formed, joining the two fragments together. At the same time, the EcoRI site on the biobrick prefix of LuxR (BBa_K1897008) and the PstI site on the biobrick suffix of GFP (BBa_K1897014) was also cut, and joined to a pSB1K3 backbone that was also cut with EcoRI and PstI. This resulted in an end product of a plasmid with LuxR and GFP joined together.

Verification of LuxR+GFP

-INSERT GEL PHOTO AS FIGURE 1-

Besides the verification of the band size via gel electrophoresis, we also verified the part by addition of external AHL. With no AHL added, fluorescence was very faint, and when 100 μm AHL was added, the fluorescence increased (Figure 2, Figure 3).

Figure 2: Induction of GFP production with/without AHL. Top: Green fluorescence microscopy images of LuxR+GFP bacteria (from left to right) of control (0 μm AHL added) and with 100 μm AHL added. Bottom: Bright field fluorescence microscopy images of LuxR+GFP bacteria (from left to right) of control (0 μm AHL added) and with 100 μm AHL added.


-INSERT GRAPH AS FIGURE 3-