Difference between revisions of "Part:BBa K349001"

Latest revision as of 17:22, 6 November 2010

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

This is the RFP BsaI 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 BsaI 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 BsaI cut sites with appropriate overhangs onto each side. For more information on these overhangs click here.

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

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 BsaI 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

The successful characterization of this part entails demonstrating its utility in the creation of new AB Bytes, because this plasmid was designed to be a base plasmid for the production of all AB bytes. The following point summarize the effectiveness of this part.

(I) RFP AB BsaI plasmid was successfully transformed, resulting in red fluorescent colonies on LB medium.

RFP AB-BsaI plasmids growing on LBO (LB medium).

(II)The RFP cassette of this plasmid was sequence-verified. It contained the proper A and B overhangs, BsaI recognition sites, BioBrick prefix and suffix and proper ORF sequences.

(III)The plasmid was cut with BsaI and showed a gel similar to that seen for the KanR plasmids

(IV)BsaI cutting efficiency and fidelity was confirmed in its use in creating other AB parts. This plasmid was used to create most AB Bytes by Team Alberta following the protocol for creating new AB bytes described above. The procedure was performed using crtE, and resulted in red and white colonies on the plate, as expected. For an indepth view of these experiments, click here.

White and red colonies produced when making parts using RFP BsaI AB Base Plasmid v.2.

(V) kan AB Bytes and the original RFP AB Byte from this plasmid were used to test the effectiveness of the assembly of multiple bytes. An anchor-KanR(AB)-Ori-RFP(AB)-Cap construct was made using the procedure described above and then it was allowed to recircularize. Numerous red fluorescent colonies were present on kanamycin LB plates the following day.

Red fluorescent colonies produced from a 5 piece Byte construction using the BioBytes 2.0 assembly method.

Together, this data demonstrates that the RFP cassette itself is functional, that the plasmid can be used to create other AB bytes, and that AB bytes made with this plasmid function properly in our multiple-Byte assembly method.

Sequence and Features