Difference between revisions of "Part:BBa K4727006:Design"
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===Design Notes=== | ===Design Notes=== | ||
| − | A strong but inducible promoter was chosen; this allows the metabolic burden to be limited, if necessary, while, when fully induced, enabling high expression levels of the alternative gene so that it can be preferentially incorporated into the phage particles. The selected promoter was pL-lac0-1 (BBa_J428041) that combines the strength of the pL promoter, with the possibility to regulate it, derived from the LacO binding site. Indeed, if the cell expresses the LacI gene at high levels, it becomes feasible to induce the expression of the gene regulated by this promoter through the addition of isopropyl β-D-1-thiogalactopyranoside (IPTG) to the culture medium. Moreover, by employing varying concentrations of the inducer, it is possible to finely adjust the expression level, thus creating a controllable system that is in line with the requirement of promoting cell vitality. | + | A strong but inducible promoter was chosen; this allows the metabolic burden to be limited, if necessary, while, when fully induced, enabling high expression levels of the alternative gene so that it can be preferentially incorporated into the phage particles. The selected promoter was pL-lac0-1 (<partinfo>BBa_J428041</partinfo>) that combines the strength of the pL promoter, with the possibility to regulate it, derived from the LacO binding site. Indeed, if the cell expresses the LacI gene at high levels, it becomes feasible to induce the expression of the gene regulated by this promoter through the addition of isopropyl β-D-1-thiogalactopyranoside (IPTG) to the culture medium. Moreover, by employing varying concentrations of the inducer, it is possible to finely adjust the expression level, thus creating a controllable system that is in line with the requirement of promoting cell vitality. |
| − | Similarly, the choice of the ribosome binding site (RBS) fell on a strong element to ensure that, upon transcript production, it is efficiently translated by ribosomes. This decision aligns with the aim of favoring the production and incorporation of this gene product over the gene 3 of phage M13. Hence, the RBS chosen was | + | Similarly, the choice of the ribosome binding site (RBS) fell on a strong element to ensure that, upon transcript production, it is efficiently translated by ribosomes. This decision aligns with the aim of favoring the production and incorporation of this gene product over the gene 3 of phage M13. Hence, the RBS chosen was <partinfo>BBa_B0034</partinfo>. |
| − | Finally, the terminator was selected based on its strength and efficiency, with a preference for an intrinsic terminator, independent from the expression of cellular proteins. Therefore, the T1 terminator of E. coli was opted (part BBa_B0010). | + | Finally, the terminator was selected based on its strength and efficiency, with a preference for an intrinsic terminator, independent from the expression of cellular proteins. Therefore, the T1 terminator of E. coli was opted (part <partinfo>BBa_B0010</partinfo>). |
===Source=== | ===Source=== | ||
Latest revision as of 07:57, 23 September 2023
M13 Gene 3 N2 deletion Cassette
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 286
Design Notes
A strong but inducible promoter was chosen; this allows the metabolic burden to be limited, if necessary, while, when fully induced, enabling high expression levels of the alternative gene so that it can be preferentially incorporated into the phage particles. The selected promoter was pL-lac0-1 (BBa_J428041) that combines the strength of the pL promoter, with the possibility to regulate it, derived from the LacO binding site. Indeed, if the cell expresses the LacI gene at high levels, it becomes feasible to induce the expression of the gene regulated by this promoter through the addition of isopropyl β-D-1-thiogalactopyranoside (IPTG) to the culture medium. Moreover, by employing varying concentrations of the inducer, it is possible to finely adjust the expression level, thus creating a controllable system that is in line with the requirement of promoting cell vitality. Similarly, the choice of the ribosome binding site (RBS) fell on a strong element to ensure that, upon transcript production, it is efficiently translated by ribosomes. This decision aligns with the aim of favoring the production and incorporation of this gene product over the gene 3 of phage M13. Hence, the RBS chosen was BBa_B0034. Finally, the terminator was selected based on its strength and efficiency, with a preference for an intrinsic terminator, independent from the expression of cellular proteins. Therefore, the T1 terminator of E. coli was opted (part BBa_B0010).
Source
M13 bacteriophage genome