Difference between revisions of "Part:BBa K4023001"
 
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glpF codes for a 28kDa<sup>1</sup> transmembrane glycerol uptake facilitator protein found in E.coli strain. glpF protein catalyze transmembrane diffusion of glycerol and some linear polyhydric alcohols in E.coli<sup>1</sup>. glpF protein also allows Arsenite to be transported into E.coli. This is as Arsenite is protonated at physiological pH and forms As(OH)3 as a major species. As(OH)3 is chemically analogous to glycerol<sup>2</sup>.
 
glpF codes for a 28kDa<sup>1</sup> transmembrane glycerol uptake facilitator protein found in E.coli strain. glpF protein catalyze transmembrane diffusion of glycerol and some linear polyhydric alcohols in E.coli<sup>1</sup>. glpF protein also allows Arsenite to be transported into E.coli. This is as Arsenite is protonated at physiological pH and forms As(OH)3 as a major species. As(OH)3 is chemically analogous to glycerol<sup>2</sup>.
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The glpF sequence was obtained from [https://parts.igem.org/Part:BBa_K190028 iGEM09_Groningen]. We optimized the sequence using IDT optimization tool and Benchling for expression within an E.coli host.
  
 
===References===
 
===References===
 
1. Fu, D., Libson, A., & Stroud, R. (2002, April). The structure of G1pF, a glycerol conducting channel. In Ion Channels: From Atomic Resolution Physiology to Functional Genomics: Novartis Foundation Symposium 245 (Vol. 245, pp. 51-65). Chichester, UK: John Wiley & Sons, Ltd.
 
1. Fu, D., Libson, A., & Stroud, R. (2002, April). The structure of G1pF, a glycerol conducting channel. In Ion Channels: From Atomic Resolution Physiology to Functional Genomics: Novartis Foundation Symposium 245 (Vol. 245, pp. 51-65). Chichester, UK: John Wiley & Sons, Ltd.
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2. Bhattacharjee, H., Mukhopadhyay, R., Thiyagarajan, S., & Rosen, B. P. (2008). Aquaglyceroporins: ancient channels for metalloids. Journal of biology, 7(9), 1-6.
 
2. Bhattacharjee, H., Mukhopadhyay, R., Thiyagarajan, S., & Rosen, B. P. (2008). Aquaglyceroporins: ancient channels for metalloids. Journal of biology, 7(9), 1-6.
  
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===Experiments and Methods===
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The experimental design can be found on our wiki in the [https://2021.igem.org/Team:Washington/Experiments Experiments]tab. Due to resitrictions imposed by the COVID 19 situation, the gene was synthesized via IDT, and transformation and verification was performed by UW Biofab. We collected the successfully transformed and streaked plates of BL21 DE3 E.coli from UW Biofab and induced protein expression by inoculating a colony of transformed E.coli in MagicMedia™ E. coli Expression Medium overnight. An aliquot of the induced bacteria were lysed for protein expression, while the rest were collected for metal tolerance assay. 
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Protein concentration of lysate was analyzed with Nanodrop, and protein concentration was diluted to 2mg/ml with 1x Laemmli buffer. The samples were then loaded into precast SDS gel and ran for ~30min at 200V. The gel was then stained with Coomassie Blue for 2 hours and destained with destaining solution, changed every 30min accompanied by gentle agitation.
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The metal tolerance assay involves the determination of the minimum inhibitory concentration of Arsenite on the successfully transformed bacteria. Briefly, initial concentration of bacteria was determined and the induced bacteria were diluted. Meanwhile LB broth containing various concentration of Sodium Arsenite solution (between 0mM to 10mM) was prepared. Subsequently, 25ul of diluted induced bacteria and 175ul of LB broth with Sodium Arsenite were added in 96 well microplates. The plates were incubated at 37 degree Celsius, and the absorbance at OD600 was taken after 20hrs of incubation.
  
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===Results===
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Due to the limited amount of time we had in the lab, the data we gathered are preliminary and requires further experimentations to improve reliability and accuracy. Nevertheless they are promising data and bodes well to the feasibility of our system design and of the intended function of the modified protein.
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<b>SDS PAGE</b>
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<br>
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The size of glpF protein is approximately 28kDa. From the gel, glpF are found in all 6 lanes, indicating that the 6 cultures of bacteria containing 6 different plasmids expressed glpF. This is expected as while only 3 E.coli cultures contained the bicistronic gene, glpF is a major intrinsic protein in E.coli and hence would be naturally to. Thus going forward, to verify the overexpression of glpF, a western blot analysis could be performed.
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[[File:T--Washington--SDS PAGE.png|400px|thumb|center|<i> Fig. 1:SDS PAGE Gel</i>]]
  
 
<!-- Add more about the biology of this part here
 
<!-- Add more about the biology of this part here

Latest revision as of 04:00, 22 October 2021


Optimized glpF

glpF codes for a 28kDa1 transmembrane glycerol uptake facilitator protein found in E.coli strain. glpF protein catalyze transmembrane diffusion of glycerol and some linear polyhydric alcohols in E.coli1. glpF protein also allows Arsenite to be transported into E.coli. This is as Arsenite is protonated at physiological pH and forms As(OH)3 as a major species. As(OH)3 is chemically analogous to glycerol2.

The glpF sequence was obtained from iGEM09_Groningen. We optimized the sequence using IDT optimization tool and Benchling for expression within an E.coli host.

References

1. Fu, D., Libson, A., & Stroud, R. (2002, April). The structure of G1pF, a glycerol conducting channel. In Ion Channels: From Atomic Resolution Physiology to Functional Genomics: Novartis Foundation Symposium 245 (Vol. 245, pp. 51-65). Chichester, UK: John Wiley & Sons, Ltd.

2. Bhattacharjee, H., Mukhopadhyay, R., Thiyagarajan, S., & Rosen, B. P. (2008). Aquaglyceroporins: ancient channels for metalloids. Journal of biology, 7(9), 1-6.

Experiments and Methods

The experimental design can be found on our wiki in the Experimentstab. Due to resitrictions imposed by the COVID 19 situation, the gene was synthesized via IDT, and transformation and verification was performed by UW Biofab. We collected the successfully transformed and streaked plates of BL21 DE3 E.coli from UW Biofab and induced protein expression by inoculating a colony of transformed E.coli in MagicMedia™ E. coli Expression Medium overnight. An aliquot of the induced bacteria were lysed for protein expression, while the rest were collected for metal tolerance assay.

Protein concentration of lysate was analyzed with Nanodrop, and protein concentration was diluted to 2mg/ml with 1x Laemmli buffer. The samples were then loaded into precast SDS gel and ran for ~30min at 200V. The gel was then stained with Coomassie Blue for 2 hours and destained with destaining solution, changed every 30min accompanied by gentle agitation.

The metal tolerance assay involves the determination of the minimum inhibitory concentration of Arsenite on the successfully transformed bacteria. Briefly, initial concentration of bacteria was determined and the induced bacteria were diluted. Meanwhile LB broth containing various concentration of Sodium Arsenite solution (between 0mM to 10mM) was prepared. Subsequently, 25ul of diluted induced bacteria and 175ul of LB broth with Sodium Arsenite were added in 96 well microplates. The plates were incubated at 37 degree Celsius, and the absorbance at OD600 was taken after 20hrs of incubation.

Results

Due to the limited amount of time we had in the lab, the data we gathered are preliminary and requires further experimentations to improve reliability and accuracy. Nevertheless they are promising data and bodes well to the feasibility of our system design and of the intended function of the modified protein.

SDS PAGE
The size of glpF protein is approximately 28kDa. From the gel, glpF are found in all 6 lanes, indicating that the 6 cultures of bacteria containing 6 different plasmids expressed glpF. This is expected as while only 3 E.coli cultures contained the bicistronic gene, glpF is a major intrinsic protein in E.coli and hence would be naturally to. Thus going forward, to verify the overexpression of glpF, a western blot analysis could be performed.

Fig. 1:SDS PAGE Gel

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
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 183