Part:BBa_K1781006

LZ-T18 - Leucine Zipper bound to T18 fragment of BACTH

Leucine zippers are one of the most common protein-binding motifs. The gene codes for an alpha helix that contains a high amount of leucine residues. This alpha helix can interact with another leucine zipper and dimerize. This interaction has been thoroughly characterized and confirmed as a good control. The leucine zipper here is bound to the T18 subunit of the BACTH system and can be used as a control for the T18 and T25 interactions.

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
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 4
  Illegal AgeI site found at 130
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI.rc site found at 49
  

Results - Conversion of BACTH into an iGEM standard and analysis of function

The inserts T25, T18, LZT18 and LZT25 all fit the iGEM Biobrick standard which meant that they had fixed prefix restriction sites and suffix restriction sites. The vectors they came in had kanamycin resistance.

Fusion ligation of T18, LZT18 and T25, LZT25

The plasmids containing T18, LZT18 and T25, LZT25 were restriction digested and gel electrophoresis was performed to purify them (figure 1).

Figure 1 - Gel run with the different plasmids. The second lane shows two bands (restriction digest of T25): the upper band is the plasmid construct and the lower band is the T25 that has been cut out. Similarly the third lane shows two bands of which the lower band is the T18 and the upper is its plasmid construct. Lanes four and five show the restriction digests of the LZT25 and LZT18, respectively. The upper bands in these lanes correspond to the vector containing LZT25 and LZT18 and the lower bands are short sequences of DNA that have been cut out to make the vector linear.

After this gel run, the necessary bands were eluted out and the inserts were ligated with their respective vectors. An electroporation was done to transform E. coli GBO5 with these plasmids and were streaked onto kanamycin resistant plates that gave colonies meaning successful transformation (figure 2).

Figure 2 - Transformed E. coli on kanamycin resistant plates. Colonies were seen in both T18/LZT18 transformed colonies (left) and T25/LZT25 transformed colonies (right).

Ligation of fusion products: T18-LZT18 and T25-LZT25

Selected colonies from the above plates were cultured to extract plasmids. These plasmids were then restriction digested and a gel electrophoresis was carried out to purify them (figure 3).

Figure 3 - Gel run with the restriction digests. The second lane shows restriction digest of T18/LZT18. There is only one band because the other DNA fragment is very small, and the band visible contains the linearized plasmid containing T18/LZT18 which acts as the vector. The third lane shows restriction digest of T25/LZT25 and the lower band contains T25/LZT25 which acts the insert.

After this gel run, the necessary bands were eluted out and the insert was ligated with the vector. An electroporation was done to transform E.coli GBO5 with these plasmids and were streaked onto kanamycin resistant plates that gave colonies meaning successful transformation (figure 4).

Figure 4 - E. coli colonies with the plasmids. The colonies seen present T18-LZT18 and T25-LZT25.

Ligation with lacZ

Selected colonies from the above plates were cultured to extract plasmids. These plasmids were then restriction digested and a gel electrophoresis was carried out to purify them (figure 5).

Figure 5 - Gel run after digesting the plasmids. The second and the fourth lanes show restriction digest of T18/LZT18 + T25/LZT25. The lower band in these lanes corresponds to the T18/LZT18 + T25/LZT25 cassette that acts as the insert and a linearized pLac Biobrick (pSB1C3 backbone) acts as the vector.

After this gel run, the necessary bands were eluted out and the insert was ligated with the vector. An electroporation was done to transform E.coli BTH101 with this plasmid and was streaked onto X-Gal plates. The plate showed several white colonies along with blue colonies which meant that the transformation was not good. This step should be repeated to achieve the hypothesised result (figure 6).

Figure 6 - X-Gal plate with blue colonies. These blue colonies correspond to E. coli BTH101 with the T18/LZT18 + T25/LZT25 plasmid. Although it cannot be properly seen, the number white colonies made the transformation unacceptable.

The vector map of the final product is given below (figure 7).

Figure 7 - Structure of the final product that the blue E. coli BTH101 colonies contain.