Part:BBa_K4195167

T7-B0034-endolysin-T7t-T7-B0034-Gam-his-T7t

Biology

Gam

Gam Nuclease Inhibitor is a recombinant protein that inhibits Exonuclease V (RecBCD) activity and stabilizes linear DNA templates in E. coli based in vitro protein synthesis reactions(1). While protecting linear DNA, it does not significantly affect expression efficiency.

Endolysin

This endolysin is from phage lambda(1). In wild-type phage lambda, three genes S, R, and Rz are responsible for lysing the host cell. Gene products R and Rz degrade the cell wall. Cells containing R and Rz gene products lyse efficiently when the inner membrane is disrupted chemically or by freezing-thawing the cells.

Usage and design

To prepare bacterial lysates for cell-free gene expression system, we build composite partBBa_K4195167, and it has been assembled into the vector pSB1C3 by standard BioBrick assembly. The constructed plasmids were transformed into E. coli BL21(DE3) in order to express endolysin.

Characterization

1. Agarose Gel Electrophoresis

We assemble BBa_K4195084, BBa_K3222000 and BBa_B0034 to obtain the composite BBa_K4195167, which are assembled on the expression vector pSB1C3 by standard assembly. The constructed plasmids are transformed into E. coli BL21(DE3), then the positive transformants were selected by chloramphenicol and confirmed by colony PCR and sequencing.

Fig. 1 The result of colony PCR. Plasmid pSB1C3.

2. Preparation and verification of cell-free gene expression system

To verify the cell-free gene expression system, the mixture of cell lysate and TX-TLpremix was split into small aliquots and frozen at −80 ℃ or used for cell-free expression directly.

Plasmids of pSB1C3 containing part BBa_K4195152(T7-GFP-T7t) and ddw of equal volume are separately added into the aliquots of cell-free sensing mix, then incubated in 30°C for 6 h (Fig. 2). The expression behavior of GFP is observed under blue-light gel imager. And the expression behavior of linear DNA is measured by comparing the Relative Fluorescence Units(RFU_GFP) between plasmid and linear DNA(Fig. 3).

Fig. 2 The verification of cell-free ribozyme-assisted sensing system using plasmid BBa_K4195152.

Fig.3 Comparison between linear DNA fragment and plasmid.

Reference

1. A. Didovyk, T. Tonooka, L. Tsimring, J. Hasty, Rapid and Scalable Preparation of Bacterial Lysates for Cell-Free Gene Expression. ACS Synth Biol 6, 2198-2208 (2017).

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 531
  Illegal NheI site found at 1121
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]