Difference between revisions of "Part:BBa K2541404"
 
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Green fluorescent protein (GFP) exhibits intrinsic fluorescence and is commonly used as a reporter gene in intact cells and organisms [1]. Many mutants of the protein with either modified spectral properties, increased fluorescence intensity, or improved folding properties have been reported [2].  
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Green fluorescent protein (GFP) exhibits intrinsic fluorescence and is commonly used as a reporter gene in intact cells and organisms <sup>[1]</sup>. Many mutants of the protein with either modified spectral properties, increased fluorescence intensity, or improved folding properties have been reported <sup>[2]</sup>.  
 
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GFP often misfolds when expressed as fusions with other proteins, while a robustly folded version of GFP, called superfolder GFP (sfGFP), was developed and described by Pédelacq et al at 2006[3] that folds well even when fused to poorly folded polypeptides. We decided to use sfGFP as our reporter protein due to its faster folded feature and higher fluorescence intensity. The superfolder GFP had been registered in iGEM BBa_I746916. However, the superfolder GFP (BBa_I746916) contains a BbsI restriction site, and the BbsI restriction endonuclease is an economical and efficient enzyme used in GoldenGate assembly, so the superfolder GFP (BBa_I746916) cannot be used for GoldenGate assembly.
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GFP often misfolds when expressed as fusions with other proteins, while a robustly folded version of GFP, called superfolder GFP (sfGFP), was developed and described by Pédelacq et al at 2006<sup>[3]</sup> that folds well even when fused to poorly folded polypeptides. We decided to use sfGFP as our reporter protein due to its faster folded feature and higher fluorescence intensity. The superfolder GFP had been registered in iGEM BBa_I746916. However, the superfolder GFP (BBa_I746916) contains a BbsI restriction site, and the BbsI restriction endonuclease is an economical and efficient enzyme used in Golden Gate assembly, so the superfolder GFP (BBa_I746916) cannot be used for Golden Gate assembly.
 
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So, this year our team improved the superfolder GFP (BBa_I746916). Firstly, we registered the superfolder GFP designed by Overkamp W et al[4] with a BBa_K2541400 (called sfGFP_optimism). It is a codon optimized sfGFP. Secondly, we made two point mutations in superfolder GFP (BBa_I746916) to eliminate its BbsI restriction site without changing its amino acid sequence. We made it from 5’-gaagac-3’ to 5’-gaggat-3’. We called the mutated variant sfGFP(BbsI free) (BBa_K2541401). The two sfGFP we designed don't have BbsI restriction site, so they can be used in GoldenGate assembly.
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So, this year our team improved the superfolder GFP (BBa_I746916). Firstly, we registered the superfolder GFP designed by Overkamp W et al<sup>[4]</sup> with a BBa_K2541400 (called sfGFP_optimism). It is a codon optimized sfGFP. Secondly, we made two site-directed mutations in superfolder GFP (BBa_I746916) to eliminate its BbsI restriction site without changing its amino acid sequence. We made it from 5’-gaagac-3’ to 5’-gaggat-3’. We called the mutated variant sfGFP(BbsI free) (BBa_K2541401). The two sfGFP we designed don't have BbsI restriction site, so they can be used in Golden Gate assembly.
 
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In order to compare the two sfGFP we designed with superfolder GFP (BBa_I746916), we designed this measurement device to measure superfolder GFP (BBa_I746916) fluorescence intensity.
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In order to compare the two sfGFP we designed with superfolder GFP (BBa_I746916), we designed this measurement device to measure sfGFP(BbsI free) (BBa_K2541401) fluorescence intensity.
 
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[[File:sfGFP_optimism f2.png|center|sfGFP_optimism f2]]
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Figure 1.
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<img src="https://static.igem.org/mediawiki/parts/5/50/SfGFP_op.svg" width="80%" />
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<img src="https://static.igem.org/mediawiki/parts/e/e3/SfGFP_BbsI_free.svg" width="80%" />
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<img src="https://static.igem.org/mediawiki/parts/1/1e/SfGFP-1.svg" width="80%" />
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Figure 1. <b>Emission and Excitation Spectra</b> of sfGFP_optimism(BBa_K2541400), sfGFP(BbsI free)(BBa_K2541401) and sfGFP(BBa_I746916).
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We detected the expression intensity of the three sfGFP. According to the results (figure 3), we found out that the fluorescence intensity of sfGFP_optimism (BBa_K2541400) and sfGFP(BbsI free) (BBa_K2541401) are higher than superfolder GFP (BBa_I746916).
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We detected the expression intensity of the three sfGFP. According to the results (Figure 2), we found out that the fluorescence intensity of sfGFP_optimism (BBa_K2541400) is higher than superfolder GFP (BBa_I746916) and sfGFP(BbsI free) (BBa_K2541401) is about the same as superfolder GFP (BBa_I746916).
 
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[[File:Jilin China-fluorescence-1.png|center|Jilin China-fluorescence-1]]
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Figure 2. <b>The expression of three types of sfGFP in E.coli.</b> The grey line represents fluorescent expression of sfGFP_optimism (BBa_K2541400), the orange line represents fluorescent expression of sfGFP(BbsI free) (BBa_K2541401), the blue line represents fluorescent expression of superfolder GFP (BBa_I746916) and the yellow line represents fluorescent expression of negtive control (BBa_J364007).
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<img src="https://static.igem.org/mediawiki/parts/8/83/Jilin_China-sfGFP-fluorescence-01.svg" width="80%" /></center></html>
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Figure 2. <b>The expression of three types of sfGFP in E.coli.</b> The grey line represents fluorescent expression of sfGFP_optimism (BBa_K2541400), the orange line represents fluorescent expression of sfGFP(BbsI free) (BBa_K2541401), the blue line represents fluorescent expression of superfolder GFP (BBa_I746916) and the yellow line represents fluorescent expression of negative control (BBa_J364007).
  
 
<h1>'''3. Conclusion'''</h1>
 
<h1>'''3. Conclusion'''</h1>
 
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We have made an improvement on the superfolder GFP (BBa_I746916). Our sfGFP_optimism (BBa_K2541400) and sfGFP(BbsI free) (BBa_K2541401) are BbsI restriction site free, which can be used
+
We have made an improvement on the superfolder GFP (BBa_I746916). Our sfGFP_optimism (BBa_K2541400) and sfGFP(BbsI free) (BBa_K2541401) are BbsI restriction site free, which can be used in Golden Gate assembly to achieve efficient and rapid assembly of gene fragments. And the fluorescence intensity of sfGFP_optimism (BBa_K2541400) is higher than superfolder GFP (BBa_I746916) and sfGFP(BbsI free) (BBa_K2541401) is about the same as  superfolder GFP (BBa_I746916).
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<span class='h3bb'>Sequence and Features</span>
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<partinfo>BBa_K2541404 SequenceAndFeatures</partinfo>
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<!-- Uncomment this to enable Functional Parameter display
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===Functional Parameters===
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<partinfo>BBa_K2541404 parameters</partinfo>
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<!-- -->

Latest revision as of 13:48, 17 October 2018


sfGFP(BbsI free) measurement device

It is a measurement device for sfGFP(BbsI free) (BBa_K2541401) fluorescence intensity, which is composed of Anderson promoter (BBa_J23104), RBS (BBa_B0034), sfGFP(BbsI free) (BBa_K2541401) and double terminator (BBa_B0010 and BBa_B0012).

1. Usage and Biology

Green fluorescent protein (GFP) exhibits intrinsic fluorescence and is commonly used as a reporter gene in intact cells and organisms [1]. Many mutants of the protein with either modified spectral properties, increased fluorescence intensity, or improved folding properties have been reported [2].

GFP often misfolds when expressed as fusions with other proteins, while a robustly folded version of GFP, called superfolder GFP (sfGFP), was developed and described by Pédelacq et al at 2006[3] that folds well even when fused to poorly folded polypeptides. We decided to use sfGFP as our reporter protein due to its faster folded feature and higher fluorescence intensity. The superfolder GFP had been registered in iGEM BBa_I746916. However, the superfolder GFP (BBa_I746916) contains a BbsI restriction site, and the BbsI restriction endonuclease is an economical and efficient enzyme used in Golden Gate assembly, so the superfolder GFP (BBa_I746916) cannot be used for Golden Gate assembly.

So, this year our team improved the superfolder GFP (BBa_I746916). Firstly, we registered the superfolder GFP designed by Overkamp W et al[4] with a BBa_K2541400 (called sfGFP_optimism). It is a codon optimized sfGFP. Secondly, we made two site-directed mutations in superfolder GFP (BBa_I746916) to eliminate its BbsI restriction site without changing its amino acid sequence. We made it from 5’-gaagac-3’ to 5’-gaggat-3’. We called the mutated variant sfGFP(BbsI free) (BBa_K2541401). The two sfGFP we designed don't have BbsI restriction site, so they can be used in Golden Gate assembly.

In order to compare the two sfGFP we designed with superfolder GFP (BBa_I746916), we designed this measurement device to measure sfGFP(BbsI free) (BBa_K2541401) fluorescence intensity.

2. Characterization

We got the emission and excitation spectra of the three sfGFP: sfGFP_optimism (BBa_K2541400), sfGFP(BbsI free) (BBa_K2541401) and superfolder GFP (BBa_I746916) (figure 2).

Figure 1. Emission and Excitation Spectra of sfGFP_optimism(BBa_K2541400), sfGFP(BbsI free)(BBa_K2541401) and sfGFP(BBa_I746916).


We detected the expression intensity of the three sfGFP. According to the results (Figure 2), we found out that the fluorescence intensity of sfGFP_optimism (BBa_K2541400) is higher than superfolder GFP (BBa_I746916) and sfGFP(BbsI free) (BBa_K2541401) is about the same as superfolder GFP (BBa_I746916).

Figure 2. The expression of three types of sfGFP in E.coli. The grey line represents fluorescent expression of sfGFP_optimism (BBa_K2541400), the orange line represents fluorescent expression of sfGFP(BbsI free) (BBa_K2541401), the blue line represents fluorescent expression of superfolder GFP (BBa_I746916) and the yellow line represents fluorescent expression of negative control (BBa_J364007).

3. Conclusion

We have made an improvement on the superfolder GFP (BBa_I746916). Our sfGFP_optimism (BBa_K2541400) and sfGFP(BbsI free) (BBa_K2541401) are BbsI restriction site free, which can be used in Golden Gate assembly to achieve efficient and rapid assembly of gene fragments. And the fluorescence intensity of sfGFP_optimism (BBa_K2541400) is higher than superfolder GFP (BBa_I746916) and sfGFP(BbsI free) (BBa_K2541401) is about the same as superfolder GFP (BBa_I746916).


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 7
    Illegal NheI site found at 30
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI.rc site found at 74