Part:BBa_K4349012

PROMOTER CP44 + RBS + NSP4 + GFP + TERMINATOR

UFMG_UFV_Brazil team designed this composite part to perform Secretion tests, in order to choose the best signal peptide to secrete the chitinases in Lactobacillus acidophilus and incorporate it in the design of our final product.

We planned to compare the fluorescence of the extracellular GFP when using different signal peptides in L. acidophilus. This construct contains signal peptide NSP4.

The construct consists of the strong constitutive promoter CP44 - that was characterized in gram-positive bacteria Lactococcus lactis-, RBS, signal peptide NSP4, GFP and terminator for Lactobacillus spp. The composite part is shown below:

Usage and Biology

UFMG_UFV_Brazil team synthesized this construct via IDT Grant, receiving it as linear double-strand DNA.

The plan was:
1- Transform E. Coli with the BBa_K439012 inside pGEM-T Easy Vector - insertion via TA cloning.
2- Insert BBa_K439012 in pSB1C3 expression vector via digestion with restriction enzymes and ligation.
3- Transform Lactobacillus acidophilus with the resultant expression plasmid (BBa_K4390012 + pSB1C3).
4- Compare the fluorescence between the different constructs with different signal peptides -> .

However, we were unable to perform the TA cloning with this composite part, because we had problems with the insertion of parts in the pGEM-T Easy cloning vector. The insertion efficiency was low, as seen in this agar plate where we tried to establish the protocol with another part + pGEM-T Easy cloning vector:

Blue/White screening: plate had many blue colonies and satellite colonies, with a few isolated white ones.

NOTE: In the Blue/White screening colonies formed by non-recombinant cells appear blue and the recombinant ones appear white.

When we tried to repeat the experiment, we obtained white colonies, as shown below:

Blue/White screening: plate had white colonies.

However, the colonies did not grow in the liquid medium and we were unable to perform the next planned steps.

To solve this problem in the future, we plan to perform TA cloning with another cloning vector.

We would like to emphasize that we think the problem is in the establishment of the TA cloning protocol, not in the design of the construct. If your team would benefit from this construct, we encourage you to try to use it and share your experiences.

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 113
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 769