Difference between revisions of "Part:BBa K3041000"
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| − | Coding gene of | + | Coding gene of suckerin-8 from the Humboldt squid <em>Dosidicus gigas</em>, codon optimized for expression in <em>E. coli</em>. |
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| + | ==Validation== | ||
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| + | The successfully synthesized suckerins, codon-optimized for <em>E.coli</em>, were PCR amplified using the standard biobrick primers. In Figure 1, the PCR products of these specific suckerins were visualized on agarose gel. Suckerin-8, shows strong aggregation of the fragments, which is probably the result of amplification of the full sequence provided by IDT. To optimize gene synthesis we ordered some proteins together, separated by a BamHI site for later separation. However, due to the use of the standard biobrick primers in the PCR reaction, it occurred in some cases that the other genes were also amplified, which was the case for suckerin-8. The correct band for suckerin-8 thus had to be gel-purified before cloning. | ||
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| + | [[File:PCR suckerin proteins.png]] | ||
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| + | <small><b>Figure 1. Amplified suckerin genes 8, 9, and 12.</b> | ||
| + | Polymerase chain reaction (PCR) products amplified with general prefix and suffix primers, shown by gel electrophoresis. Lane 1: 1kb ladder. PCR product at different concentrations of suckerin-8 (408 bp, lane 2 and 3), suckerin-9 (568 bp, lane 4 and 5), and suckerin-12 (696 bp, lane 6 and 7). The gel confirms the amplification of the desired suckerin proteins.</small> | ||
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<!-- Add more about the biology of this part here | <!-- Add more about the biology of this part here | ||
Latest revision as of 13:44, 18 October 2019
Suckerin-8
Coding gene of suckerin-8 from the Humboldt squid Dosidicus gigas, codon optimized for expression in E. coli.
Validation
The successfully synthesized suckerins, codon-optimized for E.coli, were PCR amplified using the standard biobrick primers. In Figure 1, the PCR products of these specific suckerins were visualized on agarose gel. Suckerin-8, shows strong aggregation of the fragments, which is probably the result of amplification of the full sequence provided by IDT. To optimize gene synthesis we ordered some proteins together, separated by a BamHI site for later separation. However, due to the use of the standard biobrick primers in the PCR reaction, it occurred in some cases that the other genes were also amplified, which was the case for suckerin-8. The correct band for suckerin-8 thus had to be gel-purified before cloning.
Figure 1. Amplified suckerin genes 8, 9, and 12.
Polymerase chain reaction (PCR) products amplified with general prefix and suffix primers, shown by gel electrophoresis. Lane 1: 1kb ladder. PCR product at different concentrations of suckerin-8 (408 bp, lane 2 and 3), suckerin-9 (568 bp, lane 4 and 5), and suckerin-12 (696 bp, lane 6 and 7). The gel confirms the amplification of the desired suckerin proteins.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]