Difference between revisions of "Part:BBa K2205001"
 
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<partinfo>BBa_K2205001 short</partinfo>
 
<partinfo>BBa_K2205001 short</partinfo>
  
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deGFP is a variant of Green Fluorescent Protein (GFP). It was initially designed by Siegal-Gaskins et al for expression in cell-free protein synthesis systems and is more efficiently transcribed than other variants (e.g. sfGFP).
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deGFP is a variant of Green Fluorescent Protein (GFP). It was initially designed by Shin and Noireaux (2010) for expression in cell-free protein synthesis systems and is more efficiently translated than other variants.
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Characterisation data for deGFP for collected using the expression construct <partinfo>BBa_K2205002</partinfo>
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===CFPS Expression of deGFP===
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[[File:T--Newcastle--BB_CFPS_deGFP_sfGFP.png|500px|]]
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'''CFPS Expression of ''J23100-deGFP'' and ''J23100-sfGFP'' Constructs:''' Time course for increase in fluorescence intensity of CFPS systems expressing GFP constructs over time. Each data point is an average of triplicate results, and error bars show +/- standard error. CFPS system with no DNA (Red) was used as the negative control.
  
  
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[[File:T--Newcastle--BB_deGFP.png|400px|]]
 
[[File:T--Newcastle--BB_deGFP.png|400px|]]
  
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===FACS Data - Collected by Exeter iGEM 2017 for Newcastle iGEM 2017===
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<img src="https://static.igem.org/mediawiki/2017/c/cc/T--Newcastle--BB_deGFP_FACS.png" width="600px"/>
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Figure 5: Single cell data for '''E. coli''' cells expressing deGFP (collected by Exeter iGEM). Columns 1-6: cells expressing deGFP. Columns 7 and 8: wild type Top10 cells. Red parts of the bar are non-fluorescent cells, green parts are fluorescent cells.
  
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===Usage and Biology===
 
  
 
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<span class='h3bb'>Sequence and Features</span>
 
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<partinfo>BBa_K2205001 SequenceAndFeatures</partinfo>
 
  
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===Characterization by 2019 BGU_Israel team: ===
  
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The deGFP can be expressed in a polycistronic plasmid and is expressed and translated in Serratia Marcescens. The deGFP can be used as a reporter gene in mosquito larvae, as they are transparent, and the Serratia are part of their microbiome.
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===Functional Parameters===
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<partinfo>BBa_K2205001 parameters</partinfo>
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Through integrating engineered Serratia containing a polycistronic plasmid with deGFP, we can track the spread of our engineered bacteria and assume the expression of the other genes from the polycistronic plasmid are occurring as well.
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Both figures shows the ability of deGFP to be expressed on a polycistronic plasmid in Serratia Marcescens.
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[[File:Larva GFP.png|200px|thumb|left|Larve fed with Serratia expressing Cry11Aa and deGFP – proof of deGFP expression, confocal microscope (x10)]]
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[[File:MaleGFP Gut.png|200px|thumb|middle|Male mosquito fed with Serratia expressing deGFP - proof of our bacteria integrating in the mosquito's gut microbiome. confocal microscope (x10)]]
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[[File:Cry11Aa_Western.png|200px|thumb|right|Western Blot of Bacteria expressing Cry11Aa and deGFP – proof of Cry11Aa expression]]

Latest revision as of 18:02, 10 October 2019


deGFP

deGFP is a variant of Green Fluorescent Protein (GFP). It was initially designed by Shin and Noireaux (2010) for expression in cell-free protein synthesis systems and is more efficiently translated than other variants.

Characterisation data for deGFP for collected using the expression construct BBa_K2205002


CFPS Expression of deGFP

T--Newcastle--BB CFPS deGFP sfGFP.png


CFPS Expression of J23100-deGFP and J23100-sfGFP Constructs: Time course for increase in fluorescence intensity of CFPS systems expressing GFP constructs over time. Each data point is an average of triplicate results, and error bars show +/- standard error. CFPS system with no DNA (Red) was used as the negative control.


In vivo Qualitative Comparison

T--Newcastle--BB deGFP.png

FACS Data - Collected by Exeter iGEM 2017 for Newcastle iGEM 2017

<img src="T--Newcastle--BB_deGFP_FACS.png" width="600px"/> Figure 5: Single cell data for E. coli cells expressing deGFP (collected by Exeter iGEM). Columns 1-6: cells expressing deGFP. Columns 7 and 8: wild type Top10 cells. Red parts of the bar are non-fluorescent cells, green parts are fluorescent cells.


Characterization by 2019 BGU_Israel team:

The deGFP can be expressed in a polycistronic plasmid and is expressed and translated in Serratia Marcescens. The deGFP can be used as a reporter gene in mosquito larvae, as they are transparent, and the Serratia are part of their microbiome.

Through integrating engineered Serratia containing a polycistronic plasmid with deGFP, we can track the spread of our engineered bacteria and assume the expression of the other genes from the polycistronic plasmid are occurring as well.

Both figures shows the ability of deGFP to be expressed on a polycistronic plasmid in Serratia Marcescens.

Larve fed with Serratia expressing Cry11Aa and deGFP – proof of deGFP expression, confocal microscope (x10)
Male mosquito fed with Serratia expressing deGFP - proof of our bacteria integrating in the mosquito's gut microbiome. confocal microscope (x10)
Western Blot of Bacteria expressing Cry11Aa and deGFP – proof of Cry11Aa expression