Part:BBa_K2109102:Design
lambda cl-Nb (BH-GBP3)
This part was designed as part of the positive control of our bacterial two-hybrid single-domain antibody (sdAb) selection system. It contains the previously characterized λcl-Nb fusion protein and the coding sequence of the anti-GFP sdAb (BH-GBP3). This part is under the control of a lacUV5 IPTG inducible promoter.
When we test the system, we expect to see activation of the reporter cassette when the λcl-Nb (BH-GBP3) is in the presence of the RNAPα-pBAD (GFP) fusion protein. The BH-GBP3 has been previously characterized () therefore we anticipate that it will bind only to GFP. The λcl-Nb (BH-GBP3) fusion protein will bind to the λcl DNA binding domain located upstream of the weak promoter. If the GFP domain of RNAPα-pBAD (GFP) interacts with its own sdAb, the RNAPα subunit will be near the promoter and therefore will be able to recruit the holoenzyme. This will lead to the transcription initiation of the red fluorescent protein (RFP) in the reporter cassette. This activation can be detected using fluorescent assisted cell sorting (FACS), and would allow for rapid selection between strong and weak binding events.
The λcl-Nb (BH-GBP3) was assembled using restriction digest, a XhoI cut site was introduced between the two parts to facilitate the introduction of future sdAb sequences. The sequence of the BH-GBP3 part was verified using DNA sequencing, and potential clones were selected using colony PCR.
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal XhoI site found at 795
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
Design Notes
Unfortunately, the part is not submitted yet due to the fact that it was not in pSB1C3
The synthesis of the DNA fragments was done by Integrated DNA Technologies (IDT)
References
1. Pellis et al. (2012) A bacterial-two-hybrid selection system for one-step isolation of intracellularly functional nanobodies. Arch Biochem Biophys., 526(2): 114-123.