Difference between revisions of "Part:BBa K1899003"

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Expression levels of mut3bGFP and sfGFP were compared. Both reporters were driven by a strong constitutive promoter, BBa_J23101. Results indicated that the expression levels of sfGFP was higher than that of mut3bGFP by 0.16 fold, under an unpaired t-test, the P value was <0.0001, which indicates a very significant difference.  
 
Expression levels of mut3bGFP and sfGFP were compared. Both reporters were driven by a strong constitutive promoter, BBa_J23101. Results indicated that the expression levels of sfGFP was higher than that of mut3bGFP by 0.16 fold, under an unpaired t-test, the P value was <0.0001, which indicates a very significant difference.  
  
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[[File:IGEM2016 HKUST sfGFPcharacterization.jpg|thumb|600px|center|<b>Fig 1. Comparison of the expression levels between mut3b-gfp and sfgfp (Waldo). </b> Cells were first precultured overnight and were subcultured to mid-log phase where GFP emission measurements were made using an EnVision® multilabel reader. This result was obtained by combining 3 characterization data obtained in 3 different days. Error bar present SEM from 3 biological replicates.]]
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[[File:IGEM2016 HKUST sfGFPcharacterization.jpg|thumb|600px|center|<b>Fig 1. Comparison of the expression levels between mut3b-gfp and sfgfp. </b> Cells were first precultured overnight and were subcultured to mid-log phase where GFP emission measurements were made using an EnVision® multilabel reader. This result was obtained by combining 3 characterization data obtained in 3 different days. Error bar present SEM from 3 biological replicates.]]
 
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[[File:IGEM2016 HKUST sfGFPprotein.jpg|thumb|1700px|center|<b>Fig 2. Protein Sequence comparison of sfGFP (Waldo) and Mut3bGFP.]]
 
[[File:IGEM2016 HKUST sfGFPprotein.jpg|thumb|1700px|center|<b>Fig 2. Protein Sequence comparison of sfGFP (Waldo) and Mut3bGFP.]]

Latest revision as of 10:05, 15 October 2016


Superfolder GFP

Variant of GFP that folds robustly even when fused to poorly folded proteins.


Usage and Biology

Waldo and other research groups has reported the engineering of a superfolder GFP (sfGFP) that showed increased resistance to denaturation, improved folding kinetics, and increased resistance to aggregation during refolding (Pedelacq et al., 2006; Andrews et al., 2007; Fisher and DeLisa, 2008). sfGFP has proven to be very useful as a scaffold for improved protein detection and tagging both in vivo and in vitro using self-assembled sfGFP fragments (Cabantous et al., 2005b; Cabantous and Waldo, 2006). Furthermore, sfGFP fusions are more soluble than conventional GFP fusions (Wu et al., 2009).

Results

Expression levels of mut3bGFP and sfGFP were compared. Both reporters were driven by a strong constitutive promoter, BBa_J23101. Results indicated that the expression levels of sfGFP was higher than that of mut3bGFP by 0.16 fold, under an unpaired t-test, the P value was <0.0001, which indicates a very significant difference.

Fig 1. Comparison of the expression levels between mut3b-gfp and sfgfp. Cells were first precultured overnight and were subcultured to mid-log phase where GFP emission measurements were made using an EnVision® multilabel reader. This result was obtained by combining 3 characterization data obtained in 3 different days. Error bar present SEM from 3 biological replicates.



Fig 2. Protein Sequence comparison of sfGFP (Waldo) and Mut3bGFP.



Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal XhoI site found at 440
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 741
  • 1000
    COMPATIBLE WITH RFC[1000]



Reference

1. Al-Homsi, L., Al-Assad, J. M., Kweider, M., Al-Okla, S., & Abbady, A. Q. (2012). Construction of pRSET-sfGFP plasmid for fusion-protein expression, purification and detection. Jordan J Biol Sci, 5(4), 279-288.

2. Pedelacq JD, Cabantous S, Tran T, Terwilliger TC and Waldo GS. 2006. Engineering and characterization of a superfolder green fluorescent protein. Nat Biotechnol., 24: 79-88.

3. Andrews BT, Schoenfish AR, Roy M, Waldo G and Jennings PA. 2007. The rough energy landscape of superfolder GFP is linked to the chromophore. J Mol Biol., 373: 476-490.

4. Fisher AC and DeLisa MP. 2008. Laboratory evolution of fastfolding green fluorescent protein using secretory pathway quality control. PLoS One, 3: e2351.

5. Cabantous S, Terwilliger TC and Waldo GS. 2005b. Protein tagging and detection with engineered self-assembling fragments of green fluorescent protein. Nat Biotechnol., 23: 102-107.

6. Cabantous S and Waldo G. 2006. In vivo and in vitro protein solubility assays using split GFP. Nat Methods, 3: 845-854.

7. Waldo, G. S., Standish, B. M., Berendzen, J., & Terwilliger, T. C. (1999). Rapid protein-folding assay using green fluorescent protein. Nature biotechnology, 17(7), 691-695.

8. Waldo, G. S. (2003). Improving protein folding efficiency by directed evolution using the GFP folding reporter. Directed Enzyme Evolution: Screening and Selection Methods, 343-359.