LacI BioBrick Construction

Aims

• To use PCR to extract lacI (promoter, ribosome-binding site (RBS) & coding sequence (CDS)) from plasmid pMutin4 and ligate into vector pSB1AT3 in front of red fluorescent protein (RFP).

• The aim of today's experiment is to screen for the lacI insert. This could be done using PCR but that would require specific primers and is not as reliable.

Protocol

1. Miniprep - the miniprep protocol was followed.
2. Restriction digests - the restriction digests protocol was followed to allow the length of the DNA to be checked using single and double digest.
   • Single digest with Pst1
   • double digests with Pst1 and Xba1
3. Gel electrophoresis - the protocol for gel electrophoresis was followed.
4. Set up liquid broth culture in LB. The protocol for growing an overnight culture was followed.

Results

• The single digests (lanes 1-7) and the double digests (lanes (9-15) are separated by the molecular marker (lane 8).

Figure 1: Gel electrophoresis of the single and double digests using Pst1 and Xba1.

• Lane 1:
• Lane 2:
• Lane 3:
• Lane 4:
• Lane 5:
• Lane 6:
• Lane 7:
• Lane 8: 1kb DNA ladder
• Lane 9:
• Lane 10:
• Lane 11:
• Lane 12:
• Lane 13:
• Lane 14:
• Lane 15:

Inference