Part:BBa_K349011

KanR-AB-BfuAI (for construction of AB BioBytes 2.0)

BioBytes 2.0 assembly method testing plasmid containing the kanamycin resistance cassette. Digestion with BfuAI allows for the addition of any other AB Byte to the plasmid.

Usage and Biology

This part is compatible with the Team Alberta 2010 Biobytes 2.0 assembly method. It can be utilized as an AB BioByte following digestion with BfuAI and purification. The following protocol outlines the Biobytes 2.0 assembly method:

1) Mix the iron micro beads for 10 minutes.
2) Transfer 20 ul of iron micro beads to a 1.5 mL tube.
3) Pull the beads to the side using a magnetic tube rack.
4) Remove and discard the supernatant.
5) Add 50 ul of BioBytes 2.0 Wash Buffer to the beads. Flick gently to resuspend.
6) Pull the beads to the side using a magnetic tube rack.
7) Remove and discard the supernatant.
8) Repeat steps 5 to 7.
9) Add 200 ng of a premade anchor-Byte construct (premade in the GENOMIKON kit) to the beads and top off the volume to 20ul with TE Buffer. Flick gently to resuspend.
10) Allow annealing for 30 minutes, mixing by flicking every 5 minutes. Ensure that there are no droplets on the sides of the tube.
11) Repeat steps 6 and 7.
12) Repeat steps 5 to 7.
13) Add 200 ng of the next BioByte of your construct, making sure that a BA Byte follows an AB Byte and vice versa. Add an appropriate amount of 2x QuickLigase Buffer, Quick Ligase and TE to a total volume of 20ul.
14) Flick gently to resuspend. Allow ligation for five minutes, flicking gently every minute.
15) Add 30ul of Wash Buffer to the tube. Flick gently.
16) Repeats steps 6 and 7.
17) Repeat steps 5 to 7 twice.
18) Repeat steps 13 to 17 for each subsequent Byte addition, including the last BioByte.
19) After the last Byte has been ligated and washed, add 30 ul of 75C Elution Buffer. Keep at 75C for 10 minutes.
20) After 10 minutes, still at 75C, put the tube into a magnetic rack . Allow the beads to be pulled aside and collect the supernatant into a clean 1.5 mL tube. The supernatant will contain your construct.

For more information on this construction method click here

Characterization

The successful characterization of this part entails demonstrating its utility as a selection marker and also its ability to be excised from the plasmid using its corresponding enzyme. This allows it to be useful as either a vector for the storage of other biobyte parts or for it to be used as a part in the assembly of a novel plasmid.

(I) KanR AB BfuAI plasmid was successfully transformed, resulting in colonies on LB kanamycin medium.

(II) All KanR plasmids were cut using their respective enzymes to test effectiveness of the enzymes as well as whether the cut sites in all plasmids were intact. This gel can be viewed on the KanR-AB-BsaI page.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]