Difference between revisions of "Part:BBa K1317002:Design"
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===Design Notes=== | ===Design Notes=== | ||
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− | [[File: | + | '''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. | ||
+ | |||
+ | <center>'''Synthesis of the gene coding for the SLPs'''</center> | ||
+ | |||
+ | 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'''. | ||
+ | |||
+ | <center>Table 1 : sequences of the 8 nucleotides | ||
+ | |||
+ | [[file:Bdx2014_SLP_synthesis_01.jpg]]</center> | ||
+ | |||
+ | 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. | ||
+ | |||
+ | <center>[[file:Bdx2014_SLP_synthesis_02.jpg|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''</center> | ||
+ | |||
+ | 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. | ||
+ | |||
+ | |||
+ | <center>[[File:Bdx2014 SLP synthesis03.png]] | ||
+ | |||
+ | ''Figure 2: PCR Fusion strategy to assemble the gene coding for the SLPs''</center> | ||
+ | |||
+ | 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. | ||
+ | |||
+ | <center>Table 2: Sequence of the new fragments | ||
+ | |||
+ | [[File:Bdx2014 SLP synthesis04.png]]</center> | ||
+ | |||
+ | 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. | ||
+ | |||
+ | <center>[[file:Bdx2014_slp5.png]] | ||
+ | |||
+ | ''Fig 3 : Strategy to assemble the CDS for the SLPs using the new fragments 6 and 7''</center> | ||
+ | |||
+ | This method allows the assembly of the 7 fragments (figure 4). A fragment of 318 bp was expected on the electrophoresis gel. | ||
+ | |||
+ | <center>[[File:Bdx2014 Slp6.png]]</center> | ||
+ | |||
+ | <center>''Figure 4 : Gel electrophoresis on 3% agarose''</center> | ||
+ | |||
===Source=== | ===Source=== | ||
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[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 | [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) |
Latest revision as of 17:20, 17 October 2014
CDS of silk-like protein (SLP)
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 312
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE 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.
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.
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.
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.
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.
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.
This method allows the assembly of the 7 fragments (figure 4). A fragment of 318 bp was expected on the electrophoresis gel.
Source
major ampullate spidroin 1 (MaSp1) gene from Nephila clavipes
References
[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)