BioBricks

The following constructs were generated in the pSB1AK8 vector:

• T7 promoter-RBS-eGFP-TEV-RiAFP-His-Terminator
• T7 promoter-RBS-RiAFP-His-Terminator
• T7 promoter-RBS-eGFP-ZeAFP-His-Terminator
• T7 promoter-RBS-His-eGFP-TEV-TmAFP-Terminator
• T7 promoter-RBS-His-eGFP-TmAFP-Terminator
• T7 promoter-RBS-His-MBP-TEV-TmAFP-Terminator

In the pSB1A3 vector:

• RBS-ZeAFP
• In the pSB1C3 vector (submitted to the registry, parts BBa_K652000 to BBa_K652004):
• T7-RBS-eGFP-TEV-RiAFP-His-Terminator
• T7-RBS-RiAFP-His-Terminator
• T7-RBS-His-eGFP-TEV-TmAFP-Terminator
• T7-RBS-eGFP-TmAFP-His-Terminator
• RBS-ZeAFP-Terminator

A plasmid containing the TmAFP antifreeze protein was kindly provided by the Fass lab. A plasmid containing the type III ZeAFP antifreeze protein was kindly provided by the Davies lab. The DNA sequence encoding the 12kDa RiAFP was codon-optimized for E. coli, synthesized, and generously sponsored by Integrated DNA Technologies.

All genes were PCR-amplified with primers of the following form:

• Forward primer: Biobrick prefix-RBS-20bp homology to start of gene
• Reverse primer: Reverse complement of 20bp homology to end of gene-TAA-terminator-suffix

All primer designs can be found in the protocols section.

PCR products were PCR purified and gel purified, then cut with XbaI and PstI and ligated to the pSB1AK8 plasmid (containing a T7 promoter) cut with SpeI and PstI. Successful ligation was verified by colony PCR, a double digest, and sequencing. Several antifreeze constructs were transferred from the pSB1AK8 vector to the pSB1C3 vector for submission to the registry. The pSB1C3 linearized vector and the biobricks in the pSB1AK8 vector were digested with EcoRI and PstI. The pSB1AK8 vector was CIP treated prior to ligation. Colonies were selected on chloramphenicol plates, and colony PCR and sequencing were used to verify the constructs.

Figures

Figure 1: Representation of cloning strategy
Figure 2: PCR amplification of RBS-ZeAFP construct shows correct product at 300bp. Lane 1: 100bp ladder, Lane 2: PCR of ZeAFP, Lane 3: replicate. The PCR product was digested with EcoRI and PstI and ligated to linearized pSB1A3 cut with the same enzymes.
Figure 3: Colony PCR was performed on six colonies post-ligation to screen for ones containing our insert. Lane 1: 100bp ladder, Lanes 3, 5 and 6 show amplification of our insert; these colonies were subsequently miniprepped.
Figure 4: PCR amplification of several biobrick constructs. Lane 1: 1kb ladder, Lane 2: 100bp ladder, Lane 3: RBS-eGFP-TEV-RiAFP-His-Terminator, Lane 4: RBS-RiAFP-His-Terminator, Lane 5: RBS-His-eGFP-TmAFP-Terminator, Lane 6: RBS-TmAFP-His-Terminator, Lane 7: RBS-His-eGFP-TEV-TmAFP-Terminator, Lane 8: blank, Lane 9: 1kb ladder, Lane 10: 100bp ladder.
Figure 5: Colony PCR verifying successful ligation of several biobrick constructs. Lane 1: 1kb ladder, Lanes 2-3: RBS-RiAFP-His-Terminator, Lanes 4-5: RBS-eGFP-TEV-RiAFP-His-Terminator, Lanes 6-9: RBS-His-eGFP-TEV-TmAFP-Terminator, Lanes 10-11: RBS-TmAFP-His-Terminator, Lanes 12-14: RBS-His-eGFP-TmAFP-Terminator, Lane 15: 1kb ladder
Figure 6: Restriction digest verifying successful ligation of RBS-RiAFP-His-Terminator and RBS-eGFP-TEV-RiAFP-His-Terminator. Lane 1: 100bp ladder, Lane 2: 1kb ladder, Lane 3-10 RiAFP, Lane 11-13 RiAFP-GFP, Lane 14: 100bp ladder, Lane 15: 1kb ladder.
Figure 7: Colony PCR after ligation into pSB1C3 vector. Lane 1: ladder, Lane 2: ladder, Lanes 3-5: eGFP-TmAFP, Lanes 6-8: His-TmAFP, Lanes 9-11: RiAFP, Lanes 12-15: eGFP-RiAFP
Figure 8: Colony PCR after ligation into pSB1C3 vector. Lane 1: ladder, Lanes 2-5: eGFP-TEV-TmAFP, Lanes 6-8: ZeAFP, Lanes 9-12: PCR amplification of only eGFP for subsequent cloning, Lanes 13-14: ladder <groupparts>iGEM011 Yale</groupparts>