Part:BBa_K4349009

PROMOTER CP44 + RBS + Cwh + ENDOCHI + HISTAG + TERMINATOR

UFMG_UFV_Brazil team designed this composite part to express and secrete endochitinase PCCHI44 from Pochonia chlamydosporia in Lactobacillus acidophilus. The construct consists of the strong constitutive promoter CP44 - that was characterized in gram-positive bacteria Lactococcus lactis-, RBS, signal peptide Cwh, endochitinase PCCHI44, Histidine tag(6x) 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_K439009 inside pGEM-T Easy Vector - insertion via TA cloning
2- Insert BBa_K439009 in pSB1C3 expression vector via digestion with restriction enzymes and ligation.
3- Transform Lactobacillus acidophilus with the resultant expression plasmid (BBa_K439009 + pSB1C3).

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 800
  
• 1000
  COMPATIBLE WITH RFC[1000]