Part:BBa_K349005

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

This is the RFP BfuAI AB Base Plasmid v.2 for the construction of components of Team Alberta's 2010 BioBytes 2.0 assembly method. The RFP cassette can be removed using the BfuAI enzyme and a new part may be inserted into the plasmid in order to amplify it in the AB Byte format applicable to this method.

Usage and Biology

This plasmid is a useful tool for creating AB Bytes in compliance with the BioBytes 2.0 assembly method according to the following procedure:

1) PCR the part of interest, incorporating BfuAI cut sites with appropriate overhangs onto each side. For more information on these overhangs click here.

2) Digest both this RFP BfuAI AB Base Plasmid (v.2) and the PCR product with BfuAI.

3) Ligate them together. Because the overhangs are unique, the plasmid backbone can not re-ligate without an insert. The insert can only be the new part of interest, or the RFP coding cassette that was originally in the base plasmid, leading to a total of 2 possible ligation products.

4) When transformed, the ligation products can appear red (if the original RFP cassette is reinserted) or white (if the part of interest is inserted). This provides a selection method for plasmids that have the part of interest in it.

Biobytes 2.0 Assembly Protocol:

When digested with BfuAI and purified, this plasmid can yield an AB RFP cassette. It can also be used to make other AB Bytes as per the process described above and subsequently digested with BsaI and purified to yield those particular AB Bytes. Certain Bytes can be added together sequentially using the BioBytes 2.0 assembly method. For a detailed overview of this method, click here. The following protocol summarizes the steps involved in multiple Byte assembly:

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.

Characterization

(I) RFP AB BfuAI plasmid was successfully transformed, resulting in red fluorescent colonies similar to those of BBa_K349001.

(II)The plasmid was cut with BfuAI and produced expected size fragments similar to those of BBa_K349002.

(III)BfuAI cutting efficiency and fidelity was confirmed in its use in creating other AB parts. This plasmid was used to create mWasabi, and resulted in white and red colonies on the plate, as expected. Minipreps were made from the white colonies that yielded correct size bands after BsaI digestion. For an indepth view of this experiment, visit the section Selecting Colonies with our Plasmid.

Together, this data demonstrates that the RFP cassette itself is functional, cuts correctly and that the plasmid can be used to create other AB bytes.

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]