Difference between revisions of "Part:BBa K2541401"
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__NOTOC__
 
__NOTOC__
 
<partinfo>BBa_K2541401 short</partinfo>
 
<partinfo>BBa_K2541401 short</partinfo>
 +
<h5>
 +
<P style="text-indent:2em;">
 +
It is a superfolder GFP, a robustly folded version of GFP, which is BbsI restriction site free and can be used in GoldenGate assembly.
 +
</p>
 +
</h5>
  
−
It is a superfolder GFP, a robustly folded version of GFP. It is BbsI restriction site free by mutating two bases from superfolder GFP(BBa_I746916)-from 5’-gaagac-3’ to 5’-gaggat-3’, which does not change the amino acid encoded by this sequence.
+
<h1>'''1. Usage and Biology'''</h1>
 +
<h5>
 +
<P style="text-indent:2em;">
 +
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].
 +
</p>
 +
<P style="text-indent:2em;">
 +
GFP often misfold 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 better fluorescence intensity. The superfolder GFP had been registered in iGEM BBa_I746916.
 +
</p>
 +
<P style="text-indent:2em;">
 +
However, the superfolder GFP (BBa_I746916) contains a BbsI restriction enzyme cleavage 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. This year we made two point mutations in superfolder GFP (BBa_I746916) to eliminate its BbsI restriction site without changing its amino acid sequence to make it suitable for GoldenGate assembly. We changed the sequence from 5’-gaagac-3’ to 5’-gaggat-3’. And we called the mutated  variant sfGFP(BbsI free) (BBa_K2541401).
 +
</p>
 +
[[File:BbsI free.png|center|BbsI free]]
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<center>
 +
Figure 1. BbsI restriction site and point mutation.
 +
</center>
 +
----
  
−
<h1>'''Usage and Biology'''</h1>
 
−
Green fluorescent protein (GFP) offers efficient ways of visualizing promoter activity and protein localization in vivo, and many different variants are currently available to study bacterial cell biology. It 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 misfold when expressed as fusions with other proteins, while a robustly folded version of GFP, called superfolder GFP, was developed and described by Pédelacq et al at 2006 [3]that folds well even when fused to poorly folded polypeptides. Furthermore, sfGFP might be particularly suitable for gene expression studies, since the emergence of fluorescence closely matches induction of transcription. The superfolder GFP had been registered in iGEM BBa_I746916.  
+
<h1>'''2. Characterization'''</h1>
 +
<P style="text-indent:2em;">
 +
We constrcted sfGFP_optimism (BBa_K2541400) sequence and superfolder GFP (BBa_I746916) on the pSB1C3 vector. The BbsI recognition site free was confirmed by nucleic acid electrophoresis (figure 1). The sfGFP_optimism can not be digested by BbsI. And the length of these sequences are correct.
 +
</p>
 +
[[File:sfGFP_optimism f1 new1.png|center|sfGFP_optimism f1 new1]]
 +
Figure 1. L1: 1kb DNA marker; L2: BBa_I746916; L3: BBa_I746916+BbsI; L4: BBa_K2541401; L5: BBa_K2541401+BbsI; L6: BBa_K2541400; L7: BBa_K2541401+BbsI; L8: 1kb DNA marker.  
 +
----
  
−
However, the superfolder GFP (BBa_I746916) contains a BbsI restriction enzyme cleavage 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. This year we made two point mutations in superfolder GFP (BBa_I746916) to eliminate its BbsI restriction site without changing its amino acid sequence to make it suitable for GoldenGate assembly. We made it from 5’-gaagac-3’ to 5’-gaggat-3’. We called the mutated superfolder GFP is sfGFP(BbsI free) (BBa_K2541401).
 
−
<!-- -->
 
  
−
<h1>'''Characterization'''</h1>
+
<h5>
−
The improved sfGFP(BbsI free) (K2541401) variant was standardised into BioBrick format and its fluorescence intensity was compared with superfolder GFP(BBa_I746916) .Its x fold increased fluorescence intensity is a very useful feature. And it is BbsI restriction site free, so it can be used in GoldenGate assembly to achieve efficient and rapid assembly of gene fragments.
+
<P style="text-indent:2em;">
 +
We got the emission and excitation spectra of two type sfGFP: sfGFP_optimism (BBa_K2541400) and superfolder GFP (BBa_I746916) (figure 2).
 +
</p>
 +
</h5>
 +
[[File:sfGFP_optimism f2.png|center|sfGFP_optimism f2]]
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Figure 2.
 +
----
  
  
−
The new variant sfGFP(BbsI free) (K2541401) was contributed to the Registry of Standard Biological Parts and it will be more widely used in the synthetic biology community.  
+
<h5>
 +
<P style="text-indent:2em;">
 +
For further characterization, we detected the expression intensity of these two types of sfGFP. According to the results (figure 3), we found out that the fluorescence intensity of sfGFP_optimism (BBa_K2541400) is nearly 2.6 times higher than superfolder GFP (BBa_I746916).
 +
</p>
 +
</h5>
 +
[[File:sfGFP_optimism f3.png|center|sfGFP_optimism f3]]
 +
Figure 3.
 +
----
  
  
−
<span class='h3bb'>Sequence and Features</span>
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<h1>'''3. Conclusion'''</h1>
−
<partinfo>BBa_K2541401 SequenceAndFeatures</partinfo>
+
<h5>
 +
<P style="text-indent:2em;">
 +
We have made an improvement on the superfolder GFP (BBa_I746916). Our sfGFP_optimism (BBa_K2541400) is BbsI restriction site free, which can be used in GoldenGate assembly to achieve efficient and rapid assembly of gene fragments. And its fluorescence intensity is higher than superfolder GFP (BBa_I746916).
 +
</p>
 +
</h5>
 +
  
  
 +
<span class='h3bb'>Sequence and Features</span>
 +
<partinfo>BBa_K2541401 SequenceAndFeatures</partinfo>
 
<!-- Uncomment this to enable Functional Parameter display  
 
<!-- Uncomment this to enable Functional Parameter display  
 
===Functional Parameters===
 
===Functional Parameters===
 
<partinfo>BBa_K2541401 parameters</partinfo>
 
<partinfo>BBa_K2541401 parameters</partinfo>
 
<!-- -->
 
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Revision as of 12:55, 14 October 2018


sfGFP(BbsI free)

It is a superfolder GFP, a robustly folded version of GFP, which is BbsI restriction site free and can be used in GoldenGate assembly.

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 misfold 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 better fluorescence intensity. The superfolder GFP had been registered in iGEM BBa_I746916.

However, the superfolder GFP (BBa_I746916) contains a BbsI restriction enzyme cleavage 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. This year we made two point mutations in superfolder GFP (BBa_I746916) to eliminate its BbsI restriction site without changing its amino acid sequence to make it suitable for GoldenGate assembly. We changed the sequence from 5’-gaagac-3’ to 5’-gaggat-3’. And we called the mutated variant sfGFP(BbsI free) (BBa_K2541401).

BbsI free

Figure 1. BbsI restriction site and point mutation.


2. Characterization

We constrcted sfGFP_optimism (BBa_K2541400) sequence and superfolder GFP (BBa_I746916) on the pSB1C3 vector. The BbsI recognition site free was confirmed by nucleic acid electrophoresis (figure 1). The sfGFP_optimism can not be digested by BbsI. And the length of these sequences are correct.

sfGFP_optimism f1 new1

Figure 1. L1: 1kb DNA marker; L2: BBa_I746916; L3: BBa_I746916+BbsI; L4: BBa_K2541401; L5: BBa_K2541401+BbsI; L6: BBa_K2541400; L7: BBa_K2541401+BbsI; L8: 1kb DNA marker.


<h5>

We got the emission and excitation spectra of two type sfGFP: sfGFP_optimism (BBa_K2541400) and superfolder GFP (BBa_I746916) (figure 2).

sfGFP_optimism f2

Figure 2.


For further characterization, we detected the expression intensity of these two types of sfGFP. According to the results (figure 3), we found out that the fluorescence intensity of sfGFP_optimism (BBa_K2541400) is nearly 2.6 times higher than superfolder GFP (BBa_I746916).

sfGFP_optimism f3

Figure 3.


3. Conclusion

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


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 SapI.rc site found at 13