Difference between revisions of "Part:BBa K1992003"

Latest revision as of 16:54, 21 October 2016

Tar chemoreceptor tagged with GFP

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 AgeI site found at 1282
1000
INCOMPATIBLE WITH RFC[1000]
Illegal BsaI.rc site found at 2324
Illegal SapI.rc site found at 111

Usage and Biology

E.coli native chemoreceptors cluster in the cell poles. This property is critical for signal amplification and adaptation of the cell. Although little is known about the mechanism of localization, it is important to preserve this property with our designed receptors in order to keep a functional and sensitive chemotaxis response (1).

GFP labeling is a very common way to examine the migration and localization of certain proteins in vivo. Fusion of GFP to Tar chemoreceptor enabled us to track the migration and localization of the protein to the cell poles as expected. This part is a composed from three iGEM registry BioBricks.

Design considerations

The fusion between the Tar protein and the GFP was conducted using a flexible 3XGS linker (BBa_J18921). Tar sequence was amplified from BBa_K777000, using reverse transcription PCR the stop codon in the C - terminus (tga) was replaced with Gly codon (gga) and the rest of the linker sequence was added (Fig. 1). The GFP sequence was amplified from BBa_E0040 and the linker sequence was added to the N - terminus of the protein using the same method. The two PCR products were then fused using Gibson assembly.

Fig. 1: Sequncing results, the linker sequence is marked (a) Red frame- first line present the designed sequence. second line present the sequencing result (b) Sequencing chromatogram

Experiments and results

The fused Tar-GFP protein was cloned to our expression systems (BBa_K1992008 and BBa_K1992009) in order to examine the migration of the receptor. By using fluorescence microscopy, it can be seen in figure 2, that the receptor is clustered in the poles of the bacteria as expected.

Fig2. (a) GFP expression in the cytoplam - positive control (b) Tar expression in UU1250 strain cloned with K1992004 expression system - negative control. (c) Tar-GFP expression in UU1250 strain cloned with K1992009 expression system. (d) Tar-GFP expression in UU1250 strain cloned with K1992008 expression system.

Reference

1.SHIOMI, Daisuke, et al. Helical distribution of the bacterial chemoreceptor via colocalization with the Sec protein translocation machinery. Molecular microbiology, 2006, 60.4: 894-906.‏

@@ Line 8: / Line 8: @@
 ==Usage and Biology==
-E.coli native chemoreceptors cluster in the cell poles. This property is critical for signal amplification and adaptation of the cell. Although little is known about the mechanism of localization, it is important to preserve this property with our designed receptors in order to keep a functional and sensitive chemotaxis response (1).
+<i>E.coli</i> native chemoreceptors cluster in the cell poles. This property is critical for signal amplification and adaptation of the cell. Although little is known about the mechanism of localization, it is important to preserve this property with our designed receptors in order to keep a functional and sensitive chemotaxis response (1).
 GFP labeling is a very common way to examine the migration and localization of certain proteins in vivo. Fusion of GFP to Tar chemoreceptor enabled us to track the migration and localization of the protein to the cell poles as expected. This part is a composed from three iGEM registry BioBricks.
@@ Line 14: / Line 14: @@
 ==Design considerations==
-The fusion between the Tar protein and the GFP was conducted using a flexible 3XGS linker (<partinfo>J18921</partinfo>). Tar sequence was amplified from <partinfo>K777000</partinfo>, using reverse transcription PCR the stop codon in the C - terminus (tga) was replaced with Gly codon (gga) and the rest of the linker added (fig1). The GFP sequence was amplified from <partinfo>E0040</partinfo> and the linker added to the N - terminus of the protein using the same method. The two PCR products were then coupled using Gibson assembly.
+The fusion between the Tar protein and the GFP was conducted using a flexible 3XGS linker (<partinfo>J18921</partinfo>). Tar sequence was amplified from <partinfo>K777000</partinfo>, using reverse transcription PCR the stop codon in the C - terminus (tga) was replaced with Gly codon (gga) and the rest of the linker sequence was added (Fig. 1). The GFP sequence was amplified from <partinfo>E0040</partinfo> and the linker sequence was added to the N - terminus of the protein using the same method. The two PCR products were then fused using Gibson assembly.
+[[file:T--Technion Israel--alignment_gfp.jpg|600px|thumb|center|Fig. 1: Sequncing results, the linker sequence is marked (a) Red frame- first line present the designed sequence. second line present the sequencing result  (b) Sequencing chromatogram  ]]
 ==Experiments and results==
 <p align="justify">
-The fused Tar-GFP protein was cloned to our expression systems (<partinfo>K1992008</partinfo> and <partinfo>K1992009</partinfo>) in order to examine the migration of the receptor. By using a fluorescence microscope it can be seen in figure 2 that the receptor is closter in the poles of the bacteria as expected.
+The fused Tar-GFP protein was cloned to our expression systems (<partinfo>K1992008</partinfo> and <partinfo>K1992009</partinfo>) in order to examine the migration of the receptor. By using fluorescence microscopy, it can be seen in figure 2, that the receptor is clustered in the poles of the bacteria as expected.
 <br><br><br>
 [[file:T--Technion_Israel--Tar_flourecent.png|600px|thumb|center|Fig2. (a) GFP expression in the cytoplam - positive control (b) Tar expression in UU1250 strain cloned with K1992004 expression system - negative control. (c) Tar-GFP expression in UU1250 strain cloned with K1992009 expression system. (d) Tar-GFP expression in UU1250 strain cloned with K1992008 expression system. ]]