Difference between revisions of "Part:BBa K3041017"

Latest revision as of 13:21, 19 October 2019

P_Lac-suckerin-12

Coding gene of suckerin-12 of the Humboldt squid (Dosidicus gigas), codon-optimized for production in Escherichia. coli, BBa_K3041002 [1]was placed under control of the Lac expression cassette BBa_K314103 [2]

Validation

We were able to successfully synthesize suckerin-12, codon-optimized for E.coli . The gene was PCR amplified using the standard biobrick primers. In Figure 1, the PCR products of these specific suckerins are visualized on agarose gel. Suckerin-12 is represented as thick single bands (BBa_K3041002).

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 Suckerin-12 gene was inserted into 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-12 gene. These constructs were checked by PCR with standard biobrick primers and placed on an agarose gel>. The PCR resulted in bands validating the plasmid. This plasmid called, pBS1A3-P_Lac-suck12 was then transformed into E. coli Rosetta. E.coli Rosetta colonies expressing Suckerin-12 using the pBS1A3-P_Lac-suck12 were successfully obtained, resulting in Suckerin-12 producing Rosetta strains. 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 (Fig. 2). The SDS-PAGE shows the presence of Suckerin-12, however, it should be noted that the protein mixture obtained by the protocol was not dialyzed, resulting in an impure product.

Figure 2. On SDS-PAGE gel there is visible suckerin protein purified from E. coli, for each of the different suckerin proteins.
Lane 1: ladder, Lane 2: irrelevant, Lane 3: suckerin-12 (23 kDa, lane 3).

Successful confirmation of suckerin-12 production motivated us to proceed toward scaling up the bioprocessing to larger volumes. Considering the recent publication of hydrogel formation, and its consequent repetition of modules architecture similar to suckerin-19, we selected suckerin-12 as the optimal hydrogel candidate. In order to improve protein production, the E. coli Rosetta colonies transformed with pBS1A3-P_Lac-suck12, were pre-cultured overnight prior to inoculation in 1L fermentors. After harvesting the cells, the protein was purified using inclusion body purification with dialysis and lyophilization. Figure 3 shows an SDS-PAGE of the dialyzed suckerin-12 protein mixtures, demonstrating clear bands at around 23 kDa.

Figure 3. SDS-PAGE gel of suckerin-12 after purification and dialysis.
Lane 1: Ladder, Lane 2+3: irrelevant, Lane 4+5: suckerin-12 protein mixture

In the end, we have been able to succesfully produce suckerin-12 in E.coli. After fermentation, purification and dialysis we ended up with a purified product which was clearly visible on SDS-PAGE. Unfortunately, due to low overall suckerin yield and time constraints, we were not able to produce such hydrogel.

Sequence and Features