Part:BBa_K4075008

T7-LacO Promoter+ RiboJ + RBS_0034 + OmpA signal peptide + proDRS-N1 + double Terminator

This part is made of a T7-LacO Promoter, Ribo J, RBS_0034, OmpA signal peptide, 6xHis-tag, followed by the sequence coding for the antimicrobial peptide (AMP) DRS-N1, characterized from Phyllomedusa nordestina, and ends in a double terminator. This device enables the expression of a proAMP in Escherichia coli strains with T7 polymerase.

Assembly

Plasmid Linearization

First we linearized the pBBR1MCS-2 plasmid, this was an important step as the selected site was used to generate overlap sequences. These primers can also be used at pU19 among different plasmids containing Lacz sequence.

pBBR1MCS-2/pUC19 Forward:

• 5’-TTCCCAGTCACGACGTTG

pBBR1MCS-2/pUC19 Reverse:

• 5’-CCAACTTAATCGCCTTGCAG

Figure 1: Lanes three to seven are the results from different PCR reactions to linearize the pBBR1MCS-2 plasmid. The first lane with the EcoRI digested lambda DNA worked as a ladder since the available one was impracticable. Using literature information about lambda DNA, we could estimate the molecular weight of fragments, therefore concluding the successful linearization of the plasmid for its size of 5148 bp.

Assembly and cloning

The synthesis of the device T7-LacO Promoter + RiboJ + RBS_0034 + 6xHis-tag + proDRS-N1 + double Terminator plus overlaps insert and linear pBBR1MCS-2 plasmids were incubated with Gibson Assembly Master Mix at 50°C for 1 hour. The assembly mixture was then transformed into competent E.coli DH5-alpha cells by heat shock at 42°C for 10 seconds and plated onto Luria Broth Agar plates supplemented with Kanamycin at working concentration for selection. The isolated colonies were cultivated and a miniprep was performed.

Figure 2: Overlaps designed for Gibson assembly.

A Colony PCR was performed to screen 2 colonies (G1) and also a PCR from miniprep plasmid, under the following conditions: 98°C for 40 seconds, 30X cycles of: 98°C for 10 seconds, 65°C for 30 seconds, 72°C for 35 seconds. Final extension at 72°C for 2 minutes. PCR product held at 4°. PCR products were run on a 0,8% agarose gel at 50 V for 45 minutes and visualised in a transilluminator setting. Primers used at the overlap section:

Whole insert Forward :

• 5’-GTGCTGCAAGGCGATTAAGTTG

Whole insert Reverse :

• 5’-TTACAACGTCGTGACTGGGAAC

Figure 3: The first lane with the product PCR from the BBa_K4075013 (G0 gBlock) amplification is a positive control, leading us to find the positive samples at lanes 2-3 and 9-10, from E. coli DH5-alpha, assembled with G1. Those samples were obtained by PCR amplification of miniprep and PCR colony. The difference between the positive control and positive samples is justified because G0 is 1179 bp long and G1, 614 bp.

E. coli BL21 cells were transformed with the assembly miniprep by heat shock at 42°C for 10 seconds and plated onto Luria Broth Agar plates supplemented with Kanamycin at working concentration for selection. A Colony PCR was performed to screen 4 colonies (G1)

Figure 4: The first lane is a positive control with the product PCR from the BBa_K4075013 (G0 gBlock) amplification. A linear sample of pBBR1MCS-2 was used as a negative control. At lanes four to six, colony PCR from E. coli BL21 was confirmed as positives.

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
  COMPATIBLE WITH RFC[1000]