Difference between revisions of "Part:BBa K2933103"
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===Usage and Biology=== | ===Usage and Biology=== | ||
| − | This composite part is made up with | + | This composite part is made up with 5 basic parts, the His tag, the Sumo tag, three cutting sites(NdeI, NheI and BamHI) and our target protein Elbla2-1. It encodes a protein which is Elbla2-1 fused with His-Sumo tag. The fusion protein is about 39.5kD. In order to gain the highly purified target protein, we add His-Sumo tag in N-terminal of Elbla2-1 and combine the three parts with these three cutting sites. The fusion protein can be cut off at the cutting site BamHI. It is convenient for us to purify our target protein. |
===Molecular cloning=== | ===Molecular cloning=== | ||
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'''Massive expressing:'''<br> | '''Massive expressing:'''<br> | ||
After taking samples, we transfer them into 1L LB medium and add antibiotic to 100 μg/mL final concentration. Grow them up in 37°C shaking incubator. Grow until an OD 600 nm of 0.8 to 1.2 (roughly 3-4 hours). Induce the culture to express protein by adding 0.3 mM IPTG (isopropylthiogalactoside, MW 238 g/mol) or ~0.1 gram per 1.5 liter flask. Put the liter flasks in 16°C shaking incubator for 16h. Centrifuge your bacteria in 500 mL bottles in the 4°C rotor at 4,000 RPM for 20 mins. Do this in batches until all your culture is spun down saving the cell pastes each time.<br> | After taking samples, we transfer them into 1L LB medium and add antibiotic to 100 μg/mL final concentration. Grow them up in 37°C shaking incubator. Grow until an OD 600 nm of 0.8 to 1.2 (roughly 3-4 hours). Induce the culture to express protein by adding 0.3 mM IPTG (isopropylthiogalactoside, MW 238 g/mol) or ~0.1 gram per 1.5 liter flask. Put the liter flasks in 16°C shaking incubator for 16h. Centrifuge your bacteria in 500 mL bottles in the 4°C rotor at 4,000 RPM for 20 mins. Do this in batches until all your culture is spun down saving the cell pastes each time.<br> | ||
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===References=== | ===References=== | ||
[1] Girlich D, Poirel L, Nordmann P, Diversity of naturally occurring Ambler class B metallo-β-lactamases in Erythrobacter spp. The Journal of Antimicrobial Chemotherapy [31 Jul 2012, 67(11):2661-2664] | [1] Girlich D, Poirel L, Nordmann P, Diversity of naturally occurring Ambler class B metallo-β-lactamases in Erythrobacter spp. The Journal of Antimicrobial Chemotherapy [31 Jul 2012, 67(11):2661-2664] | ||
Revision as of 11:06, 25 September 2019
His+Linker a+Sumo+Linker b+ElBlaII
This part encodes the fusion protein of His-Sumo tag and ElBla to promote the expression and purification of target protein(ElBla2-1).
Sequence and Features
Assembly Compatibility:
- 10INCOMPATIBLE WITH RFC[10]Illegal EcoRI site found at 256
- 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 256
Illegal NheI site found at 33 - 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 256
Illegal BglII site found at 145
Illegal BamHI site found at 344 - 23INCOMPATIBLE WITH RFC[23]Illegal EcoRI site found at 256
- 25INCOMPATIBLE WITH RFC[25]Illegal EcoRI site found at 256
Illegal AgeI site found at 839 - 1000COMPATIBLE WITH RFC[1000]
Usage and Biology
This composite part is made up with 5 basic parts, the His tag, the Sumo tag, three cutting sites(NdeI, NheI and BamHI) and our target protein Elbla2-1. It encodes a protein which is Elbla2-1 fused with His-Sumo tag. The fusion protein is about 39.5kD. In order to gain the highly purified target protein, we add His-Sumo tag in N-terminal of Elbla2-1 and combine the three parts with these three cutting sites. The fusion protein can be cut off at the cutting site BamHI. It is convenient for us to purify our target protein.
Molecular cloning
First, we used the vector pET-28b sumo to construct our expression plasmid. And then we converted the plasmid constructed to E. coli DH5α to expand the plasmid largely.
Figure 1. Left: The PCR result of Elbla2-1. Right: The verification results by enzyme digestion.
After verification, it was determined that the construction is successful. We converted the plasmid to E. coli BL21(DE3) for expression and purification.
Expression and purification
Pre-expression:
The bacteria were cultured in 5mL LB liquid medium with ampicillin(100 μg/mL final concentration) in 37℃ overnight.
Massive expressing:
After taking samples, we transfer them into 1L LB medium and add antibiotic to 100 μg/mL final concentration. Grow them up in 37°C shaking incubator. Grow until an OD 600 nm of 0.8 to 1.2 (roughly 3-4 hours). Induce the culture to express protein by adding 0.3 mM IPTG (isopropylthiogalactoside, MW 238 g/mol) or ~0.1 gram per 1.5 liter flask. Put the liter flasks in 16°C shaking incubator for 16h. Centrifuge your bacteria in 500 mL bottles in the 4°C rotor at 4,000 RPM for 20 mins. Do this in batches until all your culture is spun down saving the cell pastes each time.
References
[1] Girlich D, Poirel L, Nordmann P, Diversity of naturally occurring Ambler class B metallo-β-lactamases in Erythrobacter spp. The Journal of Antimicrobial Chemotherapy [31 Jul 2012, 67(11):2661-2664]