Difference between revisions of "Part:BBa K3041017"

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<partinfo>BBa_K3041017 short</partinfo>
 
<partinfo>BBa_K3041017 short</partinfo>
  
Coding gene of Suckerin-12 of the <i>Dosidicus gigas</i> (Humboldt squid) codon-optimized for production in E. coli (BBa_K3041002) under control of the Lac expression cassette (BBa_K314103)
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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==
  
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 visualised on agarose gel. Suckerin-12 is represented as single bands.  
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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:PCR suckerin proteins.png]]
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[[File:wiki3.png]]
  
 
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<small><b>Figure 1. Amplified suckerin genes 8, 9, and 12.</b><br>
<small><b>Figure 1: Amplified suckerin genes 8, 9 and 12.</b>
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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>
 
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 genes were inserted in 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 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. The bands of suckerin-12 are very faint, still, we decided to continue the transformation(fig. 2).
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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.
 
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<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:pLac.png]]
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<small><b>Figure 2: PCR of Plac-suckerin genes for plasmid validation.</b>
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Lane 1: 1kb ladder, Lane 2+3: PLac-suckerin-8, lane 4+5: PLac-suckerin-9, lane 6+7: PLac-suckerin-12</small>
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E.coli DH5a colonies expressing the suckerins on pBS1A3 plasmids under the Lac promoter were successfully obtained, resulting in the construct pBS1A3-PLac-suckerine-12. 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 7 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). 
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[[File:wiki111.png]]
  
[[File:prothis.png]]
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<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>
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Lane 1: ladder, Lane 2: irrelevant, Lane 3: suckerin-12 (23 kDa, lane 3).</small>
  
<small><b>Figure 3: On SDS-PAGE gel there is visible suckerin protein purified from E. coli, for each of the different suckerin proteins.</b>
 
Lane 1: ladder, suckerin-12 (25 kDa, lane 3), suckerin-9 (21 kDa, lane 4) and suckerin-8 (20 kDa, lane 5). Lane 2 contains the purification from suckerin produced in Saccharomyces cerevisiae [link]. 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.  </small>
 
  
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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. 
  
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[[File:wiki4.png]]
  
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-PLac-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 8 shows an SDS-PAGE of the dialyzed Suckerin-12 protein mixtures, demonstrating clear bands at around 25 kDa. It was attempted to produce a centimetre-scale hydrogel. Unfortunately, due to low overall suckerin yield and time constraints, we were not able to produce such hydrogel(Fig. 4).
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<small><b> Figure 3. SDS-PAGE gel of suckerin-12 after purification and dialysis. </b><br>
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Lane 1: Ladder, Lane 2+3: irrelevant, Lane 4+5: suckerin-12 protein mixture</small>
  
[[File:dial12.png]]
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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.
<small><b> Figure 4: SDS-PAGE gel of Suckerin-12 after purification and dialysis. </b> Lane 1: Ladder, Lane 2+3: Irrelevant, Lane 4+5: Suckerin-12 protein mixture</small>
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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).

Wiki3.png

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.

Wiki111.png

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.

Wiki4.png

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