Coding

Part:BBa_K1317002:Design

Designed by: Mounir Benkoulouche   Group: iGEM14_Bordeaux   (2014-10-07)

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 used to assemble the biobrick has been provided by GenScript even if the assembling strategy succeeded

It allows us to have the proper sequence without mutation and with sequencing results.

The strategy of our assembly is described below anyway. The gene was synthesized by ordering specific oligo with overlapping regions, and by adding a NheI site for further fusion of proteins.

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.

We firstly identified a consensus sequence, GPGQQ, GPGGY and GPGGX. We then find in literature confirmation about our consensus sequences. And we design our SLP protein as MW-[(GPGGV)2(GPGQQ)(GPGGY)]5.

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 joined 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

Thus, a new strategy was used (figure 3). The two first steps are the same than before but after this fragments 5-6 are joined to fragments 1-2-3-4 and the PCR is made with the fragments 1 and 6. Finally the fragment 7 is added and a PCR is also performed.

Bdx2014 slp5.png Fig 3 : Strategy to assemble the CDS for the SLPs using the new fragments 6 and 7

This method allows the assembly of the 7 fragments (figure 4). A fragment of 318 bp was expected on the electrophoresis gel.

Bdx2014 Slp6.png
Figure 4 : Gel electrophoresis on 3% agarose


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

[3] Michael B. Hinman, Justin A. Jones and Randolph V. Lewis. SYNTHETIC spider silk: a modular fiber. TIBTECH SEPTEMBER 2000 (Vol. 18) (PMID 10942961)