Part:BBa_K3782000

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.

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.

!!!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

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.

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.

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal PstI site found at 269
  Illegal PstI site found at 350
  Illegal PstI site found at 530
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal PstI site found at 269
  Illegal PstI site found at 350
  Illegal PstI site found at 530
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal PstI site found at 269
  Illegal PstI site found at 350
  Illegal PstI site found at 530
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal PstI site found at 269
  Illegal PstI site found at 350
  Illegal PstI site found at 530
  
• 1000
  COMPATIBLE WITH RFC[1000]

References

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