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{% block title %}Phagemid Construction{% endblock %}
{% block lead %}This page describes our work constructing a cyanobacterial “ghost phage” using Golden Gate assembly. {% endblock %}
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We decided to use a modular cloning strategy to assemble the phagemid. We elected to use the iGEM Type IIS Golden Gate Assembly standard. There were two reasons for this choice: first, it may be necessary to alter the order of the transcriptional units, and second, it will likely be necessary to optimize the use of promoter/RBS sequences to ensure adequate and appropriate expression levels. The latter is a concern because we have limited information as to the stoichiometry of the S-TIP37 capsid components. To estimate the stoichiometry of the capsid components, we again used the T7 phage as a reference. Previous studies have indicated that a significant number of capsid and internal core molecules will be required, a moderate number (less than 20) of head-tail connector and tail tubular A and B subunits. Only 6 tail-fiber like proteins will be required for each phage, along with an unknown number of capsid assembly proteins (1).
To create the phagemid, we began by designing the basic parts needed to create the S-TIP37 capsid. We obtained the gene sequences for the capsid protein, head-tail connector, capsid assembly protein, tail tubular protein A and B, internal core protein, and the tail fiber-like protein from S-TIP37 from NCBI, as described on our Project Design page. We also designed a version of the capsid protein modified by the addition of a linker and a biotin tag for a partnership with Team MSP-Maastricht (Figure 1). More details on the design of the tagged capsid protein can be found on our Partnership page.
Part Number | Strength compared to consensus |
Parts Sequence |
---|---|---|
BBa_R0085 | 1 | TAA TAC GAC TCA CTA TAG GGA GA |
BBa_R0180 | 0.72 | TTA TAC GAC TCA CTA TAG GGA GA |
BBa_R0181 | 0.5 | GAA TAC GAC TCA CTA TAG GGA GA |
BBa_R0182 | 0.3 | TAA TAC GTC TCA CTA TAG GGA GA |
BBa_R0183 | 0.09 | TCA TAC GAC TCA CTA TAG GGA GA |
Level 1 composite | Promoter | RBS | CDS | Terminator |
---|---|---|---|---|
BBa_K4268014 | BBa_R0085 | BBa_B0034 | Internal core protein BBa_4268014 |
Bba_B0015 |
BBa_K4268008 | BBa_R0181 | BBa_B0034 | Head-tail connector BBa_4268000 |
Bba_B0015 |
BBa_K4268012 | BBa_R0181 | BBa_B0034 | Tail tubular protein A BBa_4268004 |
Bba_B0015 |
BBa_K4268013 | BBa_R0181 | BBa_B0034 | Tail tubular protein A BBa_4268005 |
Bba_B0015 |
BBa_K4268009 | BBa_R0181 | BBa_B0034 | Capsid assembly protein BBa_4268001 |
Bba_B0015 |
BBa_K4268015 | BBa_R0183 | BBa_B0034 | Tail fiber-like BBa_4268007 |
Bba_B0015 |