Difference between revisions of "Part:BBa K3782000"
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<h3>Cloning</h3> | <h3>Cloning</h3> | ||
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| + | [[File:T--UNILausanne--FfIBP pCold pET.png|300px|thumb|left|'''Figure 1:''' (a) FfIBP in the FfIBP-pColdI construct was visualized by a 1% Agarose Gel. Left to right: L - 1 kb DNA Ladder (N3232), 1 – FfIBP colony PCR with pColdI-FPrimer and pColdI-RPrimer. (b) FfIBP in the pET-17b construct was visualized by a 1% Agarose Gel. Left to right: L - 1 kb DNA Ladder (N3232), 2 – FfIBP colony PCR with pET17b-FPrimer and pET17b-RPrimer.]] | ||
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FfIBP was cloned into the pET-17b plasmid using restriction and ligation via HindIII and XhoI and Gibson Assembly. The vector includes an ampicillin resistance gene and a <i>lac</i> operator, which allows for IPTG induced expression. The final construct was checked with a 1% Agarose gel (Fig.1) and via sequencing. | FfIBP was cloned into the pET-17b plasmid using restriction and ligation via HindIII and XhoI and Gibson Assembly. The vector includes an ampicillin resistance gene and a <i>lac</i> operator, which allows for IPTG induced expression. The final construct was checked with a 1% Agarose gel (Fig.1) and via sequencing. | ||
FfIBP was cloned into the pColdI plasmid using restriction and ligation via NdeI and XhoI and Gibson Assembly. The vector includes an ampicillin resistance gene and a <i>lac</i> operator, which allows for IPTG induced expression. The final construct was checked with a 1% Agarose gel (Fig.1) and via sequencing. | FfIBP was cloned into the pColdI plasmid using restriction and ligation via NdeI and XhoI and Gibson Assembly. The vector includes an ampicillin resistance gene and a <i>lac</i> operator, which allows for IPTG induced expression. The final construct was checked with a 1% Agarose gel (Fig.1) and via sequencing. | ||
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Revision as of 14:18, 9 October 2021
Flavobacterium frigoris strain PS1 ice-binding protein gene
FfIBP
FfIBP is a protein coding region that codes for an ice binding protein (IBP). IBPs, or more specifically antifreeze proteins (AFP), can bind to ice crystals and thereby prevent further ice growth. They are produced by organisms to survive in extremely cold environments. Activities of AFPs can be characterized by their thermal hysteresis (TH) or by their ice recrystallization inhibition (IRI). TH activity corresponds to the lowering of the freezing point without changing the melting point of a solution. IRI activity inhibits the growth of large ice crystals at the expense of smaller ones. The combination of these activities, which vary depending on the protein structure, prevents the freezing of body fluids and cell damage in organisms that live in environments with extremely cold temperatures.
Contents
Profile
| Name | FfIBP |
| Base pairs | 744 |
| Number of amino acids | 247 |
| Molecular weight | 25.35 kDa |
| Origin | Flavobacterium frigoris strain PS1, synthetic |
Usage and Biology
The FfIBP protein coding region was used in the following composite parts (add links). It was expressed in E. coli strain BL21 (DE3) and purified. Various tests and assays were performed to characterize and verify the functionality of this ice-binding protein. FfIBP has moderate TH and IRI activity, and it can bind to ice crystals and inhibit their growth. The aim of our project was to use it on its own or in a mixture of other antifreeze proteins to develop a solution which could be applied on sensitive plant tissues and thereby protect crops from frost damage.
FfIBP is naturally produced by the Gram-negative Antarctic bacterium Flavobacterium frigoris PS1. Its TH activity is around 2.5 K at 50 µM[1].
!!!ADD Biological context and overall mechanism of the part!!!
Characterization
To reduce frost damage during late spring freeze, we wanted to develop a solution containing antifreeze proteins, which bind to ice crystals and inhibit their growth. FfIBP was therefore chosen as one of three AFPs, cloned and expressed in E. coli BL21 (DE3), and finally purified using a His-tag affinity column and gel filtration.
Two different vectors were used for cloning: pET-17b and pColdI, containing a T7 promoter and a cold-shock protein A (cspA) promoter respectively.
Cloning
FfIBP was cloned into the pET-17b plasmid using restriction and ligation via HindIII and XhoI and Gibson Assembly. The vector includes an ampicillin resistance gene and a lac operator, which allows for IPTG induced expression. The final construct was checked with a 1% Agarose gel (Fig.1) and via sequencing. FfIBP was cloned into the pColdI plasmid using restriction and ligation via NdeI and XhoI and Gibson Assembly. The vector includes an ampicillin resistance gene and a lac operator, which allows for IPTG induced expression. The final construct was checked with a 1% Agarose gel (Fig.1) and via sequencing.
Expression and Purification
ISF Assay
FDT Assay
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal PstI site found at 269
Illegal PstI site found at 350
Illegal PstI site found at 530 - 12INCOMPATIBLE WITH RFC[12]Illegal PstI site found at 269
Illegal PstI site found at 350
Illegal PstI site found at 530 - 21COMPATIBLE WITH RFC[21]
- 23INCOMPATIBLE WITH RFC[23]Illegal PstI site found at 269
Illegal PstI site found at 350
Illegal PstI site found at 530 - 25INCOMPATIBLE WITH RFC[25]Illegal PstI site found at 269
Illegal PstI site found at 350
Illegal PstI site found at 530 - 1000COMPATIBLE WITH RFC[1000]
References
Template:Reflist- ↑ Do H, Lee JH, Lee SG, Kim HJ. Crystallization and preliminary X-ray crystallographic analysis of an ice-binding protein (FfIBP) from Flavobacterium frigoris PS1. Acta Crystallogr Sect F Struct Biol Cryst Commun [Internet]. 2012 Jul [cited 2021 Jul 7];68(Pt 7):806. Available from: /pmc/articles/PMC3388927/