Composite

Part:BBa_K3191302

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


pBAD + GarKS + NSP4

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. The NSP4 secretion peptide is attached to the N-terminus of each GarKS peptide. 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.

University of Nebraska-Lincoln 2019

To characterize BBa_K3191302, we are testing the concentration of our garvicin part through Bradford analysis and comparing its concentration to a negative control, garvicin alone, and garvicin with the NSP4 secretion tag. We expected that garvicin with the NSP4 should be able to be secreted from the cell via the Sec-dependent pathway, increasing the concentration of garvicin outside the cell.

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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.

The results of the Mini-Protean Tris-Tricine gel, which separates proteins by size similar to SDS-PAGE, revealed that all three samples might contain some level of protein contamination. This will interfere with the identification of the target protein. While there are bands present at the size that corresponds to the garvicin KS peptide, the blur associated with possible contamination of the sample interferes with the identification. Mass spectrometry analysis can be conducted on the protein sample to more conclusively identify the molecular weight of the protein.


Usage and Biology

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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