Difference between revisions of "Part:BBa K349007"

Latest revision as of 18:04, 6 November 2010

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

This is the kanR BsaI AB Base Plasmid v.1 for the construction of components of Team Alberta's 2010 BioBytes 2.0 assembly method. The kanR 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. Version 2 of the base plasmid can be found here.

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. This part is used to create AB parts for the BioBytes 2.0 assembly method.

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.

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 BsaI 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. All lanes show a 1Kb segment which is the KanR gene being seperated from the 2Kb backbone which can also be seen.

The gel shows data in the following order from left to right. Lane 1: 1 Kb ladder. Lane 2: KanR-AB-BfuAI plasmid cut with BfuAI. Lane 3: KanR-BA-BfuAI plasmid cut with BfuAI. Lane 4: KanR-AB-BsaI cut with BsaI. Lane 5: KanR-BA-BsaI cut. with BsaI. Lane 6: KanR-AB-BbsI cut with BbsI. Lane 7: KanR-BA-BbsI cut with BbsI.

(III)The kan AB Bytes from version 1 and the RFP AB Bytes from version 2 of 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.

Sequence and Features