Part:BBa_K349117

tetR-AB (BioBytes 2.0)

Tetracycline resistance cassette in the AB format of BioBytes 2.0.

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 BsaI 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

This part was made through the use of the BBa_K349001 base plasmid and the following procedure:

1) PCR of tetR, incorporating BsaI cut sites with appropriate AB 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 AB tetR 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 AB tetR is inserted).

5) Minipreps from the white colonies were digested with BsaI and showed proper-sized bands. Please note that a slightly altered base plasmid was used (pSB1A3 instead of a pSB1C3 backbone0. There is actually a BsaI cutsite within the ampR gene of pSB1A3. As a result, 3 bands are seen after digestion rather than two. The ~1200bp band represent tetR AB.

Digestion of tetR AB in pSB1A3 backbone yields three fragments due to BsaI cutsite in ampR of the backbone.

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
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 1
  Illegal BsaI.rc site found at 1300