Difference between revisions of "Part:BBa K1317002:Design"

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<center>'''Synthesis of the gene coding for the SLPs'''</center>
 
<center>'''Synthesis of the gene coding for the SLPs'''</center>
  
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We tried to assemble the gene coding for the SLPs from 8 oligonucleotids with homolog regions with the Gibson Assembly.
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We tried to assemble the gene coding for the SLPs from 8 nucleotides with homologous regions with the Gibson Assembly.
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First of all, consensus sequences for the spider silk were identified and our own protein was designed. Then, the nucleotidic sequence using the peptidic sequence was determined. We had to pay attention because our proteic sequence is made of repeted motifs.
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First of all, consensus sequences for the spider silk were identified and our own protein was designed. Then, the nucleotide sequence using the peptide sequence was determined. We had to pay attention because our protein sequence is made of repeated motifs.
  
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<center>Table 1 : sequences of the 8 nucleotides</center>
 
[[file:Bdx2014_SLP_synthesis_01.jpg]]
 
[[file:Bdx2014_SLP_synthesis_01.jpg]]
  
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[[file:Bdx2014_SLP_synthesis_02.jpg|Figure 1: Strategy of the Gibson Assembly to assemble the gene coding for the SLPs]]
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2 different methods were used with the Gibson Assembly1: in one step at 50°C or with cycles of denaturation at 95°C and annealing at 50°C (figure 1). The enzyme used was the Phusion® High Fidelity Polymerase.
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<center>[[file:Bdx2014_SLP_synthesis_02.jpg|Figure 1: Strategy of the Gibson Assembly to assemble the gene coding for the SLPs]]
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''Figure 1: Strategy of the Gibson Assembly to assemble the gene coding for the SLPs''</center>
  
 
Our 8 oligos were not properly assembled with these 2 methods, so another method was used : the PCR-Fusion.
 
Our 8 oligos were not properly assembled with these 2 methods, so another method was used : the PCR-Fusion.
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<center>[[File:Bdx2014 SLP synthesis03.png]]
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''Figure 2: PCR Fusion strategy to assemble the gene coding for the SLPs''</center>
  
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This method was not successful because fragments 6 and 7 were unable to join together. Therefore, new fragments were designed with another homologous region. The fragment 8 that added only 2 nucleotides was suppressed and these 2 nucleotides were added on fragment 7.
  
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<center>Table 2: Sequence of the new fragments
  
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[[File:Bdx2014 SLP synthesis04.png]]</center>
  
 
===Source===
 
===Source===

Revision as of 19:49, 16 October 2014

CDS of silk-like protein (SLP)


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 312
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


Design Notes

The gene was synthesized by ordering specific oligo with overlapping regions, and by adding a NheI site for the assembling.

Synthesis of the gene coding for the SLPs

We tried to assemble the gene coding for the SLPs from 8 nucleotides with homologous regions with the Gibson Assembly. First of all, consensus sequences for the spider silk were identified and our own protein was designed. Then, the nucleotide sequence using the peptide sequence was determined. We had to pay attention because our protein sequence is made of repeated motifs.

Table 1 : sequences of the 8 nucleotides

Bdx2014 SLP synthesis 01.jpg

2 different methods were used with the Gibson Assembly1: in one step at 50°C or with cycles of denaturation at 95°C and annealing at 50°C (figure 1). The enzyme used was the Phusion® High Fidelity Polymerase.

Figure 1: Strategy of the Gibson Assembly to assemble the gene coding for the SLPs Figure 1: Strategy of the Gibson Assembly to assemble the gene coding for the SLPs

Our 8 oligos were not properly assembled with these 2 methods, so another method was used : the PCR-Fusion. This method consists of different steps using the Phusion® High Fidelity Polymerase (figure 2). In a first step fragments were joining two by two, then fragments 1-2 were joined to fragments 3-4 and a PCR is achieved using fragments 1 and 4 as primers. The same method was used for fragments 5-6 and 7-8. Finally, fragments 1-2-3-4 were assembled to fragments 5-6-7-8 and a PCR was also achieved using the fragments 1 and 8 as primers.


Bdx2014 SLP synthesis03.png Figure 2: PCR Fusion strategy to assemble the gene coding for the SLPs

This method was not successful because fragments 6 and 7 were unable to join together. Therefore, new fragments were designed with another homologous region. The fragment 8 that added only 2 nucleotides was suppressed and these 2 nucleotides were added on fragment 7.

Table 2: Sequence of the new fragments Bdx2014 SLP synthesis04.png

Source

major ampullate spidroin 1 (MaSp1) gene from Nephila clavipes

References

[1] https://www.neb.com/tools-and-resources/feature-articles/gibson-assembly-building-a-synthetic-biology-toolset

[2] Shevchuk N.A. et al. Construction of long DNA molecules using long PCR-based fusion of several fragments simultaneously (2004) Nucleic Acids Res., 32(2), 19