Difference between revisions of "Part:BBa K3041015"

Latest revision as of 14:16, 18 October 2019

P_Lac-suckerin-8

Coding gene of suckerin-8 of the Humboldt squid Dosidicus gigas, codon-optimized for production in E. coli (BBa_K3041000) under control of the Lac expression cassette (BBa_K314103)

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.

The corrected genes were inserted in the pBS1A3 plasmid. After successful transformation, colonies were selected and stored in liquid stock. After checking the plasmids, the P_Lac expression cassette was placed in front of the suckerin genes. These constructs were checked by PCR with standard biobrick primers, placed on an agarose gel and transformed into E. coli. The agarose gel in Figure 6 shows the fragments of all three different suckerins together with the Lac promoter (Fig. 2).

Figure 2. PCR of P_Lac-suckerin genes for plasmid validation. Lane 1: 1kb ladder, Lane 2+3: P_Lac-suckerin-8, lane 4+5: P_Lac-suckerin-9, lane 6+7: P_Lac-suckerin-12.

E.coli DH5α colonies expressing the suckerins on pBS1A3 plasmids under the Lac promoter were successfully obtained, resulting in the construct pBS1A3-P_Lac-suckerin-8. These strains were used for initial protein production. Small scale 100 mL flask cultures were induced at an OD₆₀₀ of 0.6-0.8 with 1 mM IPTG. After harvesting, the proteins were purified using the inclusion body purification protocol since these constructs did not contain a His₆-tag. Figure 3 visualizes the successful production of the three suckerin types on SDS-PAGE. Note, the protein mixture obtained by the protocol was not dialyzed, resulting in an impure product (Fig. 3).

Figure 3. On SDS-PAGE gel there is visible suckerin protein purified from E. coli, for each of the different suckerin proteins. Lane 1: ladder, suckerin-12 (25 kDa, lane 3), suckerin-9 (21 kDa, lane 4) and suckerin-8 (20 kDa, lane 5).

Sequence and Features