Difference between revisions of "Part:BBa K777000:Experience"

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<partinfo>BBa_K777000 AddReview number</partinfo>
 
 
<I>Technion team 2016</I>
 
<I>Technion team 2016</I>
  
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<partinfo>BBa_K1992003 short</partinfo>
  
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GFP labeling is a very common way to examine the migration and location of certain proteins inside a living cell. here you can see such labeling in order to examine the migration of Tar chemoreceptor to the poles of E.coli bacteria.This property is critical for signal amplification and adaptation of the cell.
 
  
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design
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==Usage and Biology==
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this part is a composite of 3 parts from the iGEM registry. the Tar receptor (K777000) and GFP(B0040) were taken were amplified from the kit and the flexible linker (J18921) was add by using reverse transcription PCR.
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experiments and results
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<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).
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the fused Tar-GFP protein was cloned to our expression systems (K1992008,K1992009) in order to examine the migration of the receptor.  By using a fluorescence microscope it can be seen in figure 1 that the receptor is closter in the poles of the bacteria as expected.  
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Figure 1
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Fig1. (a) Tar-GFP expression in UU1250 strain cloned with K1992008 expression system. (b) Tar expression in UU1250 strain cloned with K1992004 expression system.
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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.
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==Design considerations==
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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.
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[[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  ]]
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==Experiments and results==
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<p align="justify">
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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.
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<br><br><br>
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[[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. ]]
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</p>
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<br>
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===Reference===
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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.‏
  
  
 
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Latest revision as of 06:50, 22 October 2016


This experience page is provided so that any user may enter their experience using this part.
Please enter how you used this part and how it worked out.

Applications of BBa_K777000

User Reviews

UNIQa346d65dd6f3b25e-partinfo-00000000-QINU

Technion team 2016

Tar chemoreceptor tagged with GFP


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.‏


UNIQa346d65dd6f3b25e-partinfo-00000007-QINU