Part:BBa_K4349010

PROMOTER CP44 + RBS + optimized SacB + GFP + TERMINATOR

UFMG_UFV_Brazil team designed this composite part to achieve the criteria of Part Improvement.

We performed codon optimization in the signal peptide SacB from Bacillus subtilis to use it in the gram-positive bacteria Lactobacillus acidophilus. We planned to compare the fluorescence of the extracellular GFP when using a construct with the original SacB and the optimized SacB in L. acidophilus. This construct contains the optimized SacB.

The construct consists of the strong constitutive promoter CP44 - that was characterized in gram-positive bacteria Lactococcus lactis-, RBS, optimized signal peptide SacB, 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_K439010 inside pGEM-T Easy Vector - insertion via TA cloning.
2- Insert BBa_K439010 in pSB1C3 expression vector via digestion with restriction enzymes and ligation.
3- Transform Lactobacillus acidophilus with the resultant expression plasmid (BBa_K4390010 + pSB1C3).
4- Compare the fluorescence between the original SacB and the optimized one.

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
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 796