Part:BBa_K3041017

P_Lac-suckerin-12

Coding gene of suckerin-12 of the Humboldt squid Dosidicus gigas, codon-optimized for production in E. 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 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, placed on an agarose gel and transformed into E. coli. The PCR resulted in bands validating the plasmid. The plasmid was then transformed into E. coli Rosetta. E.coli Rosetta colonies expressing the suckerins on pBS1A3 plasmids under the Lac promoter were successfully obtained, resulting in the construct pBS1A3-P_Lac-suck12. 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. It should be noted that the protein mixture obtained by the protocol was not dialyzed, resulting in an impure product (Fig. 2).

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 [4], 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 were transformed with pBS1A3-P_Lac-suck12 and pre-cultured overnight prior to inoculation. 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. It was attempted to produce a centimeter-scale hydrogel.

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

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 126
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 2265