Composite

Part:BBa_K3191301

Designed by: Alexander Meyer   Group: iGEM19_UNebraska-Lincoln   (2019-10-16)


pBAD + GarKS

Garvicin KS (GarKS) is a leaderless bacteriocin originally isolated from Lactococcus garvieae KS1546. GarKS possesses a large spectrum of antimicrobial activity against Gram-positive genera such as Staphylococcus, Listeria, Bacillus, and Enterococcus. GarKS is composed of three peptides that are 32-34 amino acids in length. Each peptide is encoded by one of three genes located next to one another: gakA, gakB, and gakC.

This part contains gakA, gakB, and gakC for the production of the complete GarKS bacteriocin. These genes are under the influence of a pBAD promoter and separated by ribosome binding sites. Both T1 and T7TE terminators exist at the end of the sequence.

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Figure 1. Protein concentration obtained from cell supernatant measured in Bradford assay.

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Figure 2. Protein sample measured in Tris-Tricine gel. The red bar indicates the expected location of garvicin KS.

The concentrations of protein collected from a Bradford assay can be seen in figure 1. The negative control, pSB3K3, had the lowest noted concentration of 0.335 mg/mL. This indicates the basal level of protein production in the cells. The culture containing the garvicin KS gene displayed a protein concentration of 0.528 mg/mL, indicating an increase in the concentration of protein produced and excreted from the cell. The culture expressing garvicin with the NSP4 secretion peptide demonstrated a concentration of >1.044 mg/mL. This suggests that the inclusion of the secretion peptide successfully increased protein secretion.


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 125
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 65
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


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Parameters
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