Difference between revisions of "Part:BBa K3041017"
(29 intermediate revisions by 3 users not shown) | |||
Line 3: | Line 3: | ||
<partinfo>BBa_K3041017 short</partinfo> | <partinfo>BBa_K3041017 short</partinfo> | ||
− | Coding gene of | + | Coding gene of suckerin-12 of the Humboldt squid (<i>Dosidicus gigas</i>), codon-optimized for production in <i>Escherichia. coli</i>, BBa_K3041002 [https://parts.igem.org/Part:BBa_K3041002]was placed under control of the Lac expression cassette BBa_K314103 [https://parts.igem.org/Part:BBa_K314103] |
==Validation== | ==Validation== | ||
− | + | We were able to successfully synthesize suckerin-12, codon-optimized for <em>E.coli </em>. 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). | |
− | [[File: | + | [[File:wiki3.png]] |
+ | <small><b>Figure 1. Amplified suckerin genes 8, 9, and 12.</b><br> | ||
+ | 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> | ||
− | |||
− | |||
+ | 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<sub>Lac</sub> 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<sub>Lac</sub>-suck12 was then transformed into <em> E. coli</em> Rosetta. | ||
+ | <em>E.coli</em> Rosetta colonies expressing Suckerin-12 using the pBS1A3-P<sub>Lac</sub>-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 OD<sub>600</sub> 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 His<sub>6</sub>-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. | ||
− | + | [[File:wiki111.png]] | |
− | + | <small><b>Figure 2. On SDS-PAGE gel there is visible suckerin protein purified from <em>E. coli</em>, for each of the different suckerin proteins.</b><br> | |
+ | Lane 1: ladder, Lane 2: irrelevant, Lane 3: suckerin-12 (23 kDa, lane 3).</small> | ||
− | |||
− | |||
− | + | 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 <em>E. coli</em> Rosetta colonies transformed with pBS1A3-P<sub>Lac</sub>-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. | |
− | [[File: | + | [[File:wiki4.png]] |
− | <small><b>Figure 3 | + | <small><b> Figure 3. SDS-PAGE gel of suckerin-12 after purification and dialysis. </b><br> |
− | Lane 1: | + | Lane 1: Ladder, Lane 2+3: irrelevant, Lane 4+5: suckerin-12 protein mixture</small> |
+ | In the end, we have been able to succesfully produce suckerin-12 in <i>E.coli</i>. 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. | ||
− | |||
− | |||
− | |||
− | |||
− | |||
Latest revision as of 13:21, 19 October 2019
PLac-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 PLac 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-PLac-suck12 was then transformed into E. coli Rosetta.
E.coli Rosetta colonies expressing Suckerin-12 using the pBS1A3-PLac-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-PLac-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
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 126
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 2265