Part:BBa_K3041017

P_Lac-suckerin-12

Coding gene of Suckerin-12 of the Dosidicus gigas (Humboldt squid) codon-optimized for production in E. coli (BBa_K3041002) 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 are visualized on agarose gel. Suckerin-12 is represented as single bands.

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 2 shows the fragments of all three different suckerins together with the Lac promoter. Although, the bands of suckerin-12 are very faint, we decided to continue the transformation.

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 DH5a 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. 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 (23 kDa, lane 3), suckerin-9 (19 kDa, lane 4) and suckerin-8 (17 kDa, lane 5). Lane 2 contains the purification from suckerin produced in Saccharomyces cerevisiae. However, the protein was purified with a protocol optimized for bacteria resulting in the purification high amount of non-specific proteins, making these bands largely inconclusive.

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 4 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. Unfortunately, due to low overall suckerin yield and time constraints, we were not able to produce such hydrogel (Fig. 4).

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

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