Team:NTU Taida/Circuit2

Here are three kinds of signal sequence:

1.PelB (66mer) ATG AAA TAT CTG CTG CCG ACC GCG GCG GCG GGC CTG CTG CTG CTG GCG GCG CAG CCG GCG ATG GCG

2.OmpA (63mer) ATG AAA AAA ACC GCG ATT GCG ATT GCG GTG GCG CTG GCG GGC TTT GCG ACC GTG GCG CAG GCG

3.PhoA (63mer) ATG AAA CAG AGC ACC ATT GCG CTG GCG CTG CTG CCG CTG CTG TTT ACC CCG GTG ACC AAA GCG

Unlike others, we have our own way to synthesize the signal sequence.

It’s common to order whole gene sequences to Biotech, but the cost is always shocking, and since the budget limitation, we strive to find our way out.

Here is our strategy:

First, we ordered 4 primers which can combine to each other, and put them into the plasmid backbone.

Second, we link the iGEM’s official prefix and suffix (EcoRI, XbaI, SpeI and PstI) to our signal sequence, and choose XbaI、SpeI as restriction sites.

Illustrations can help you understand:

1.PelB (66mer)

GAATTC GCGGCCGC T TCTAG ATG AAA TAT CTG CTG CCG ACC GCG GCG GCG GGC CTG CTG CTG CTG GCG GCG CAG CCG GCG ATG GCG T ACTAGT A GCGGCCG CTGCAG

CTTAAG CGCCGGCG A AGATC TAC TTT ATA GAC GAC GGC TGG CGC CGC CGC CGC GAC GAC GAC GAC CGC CGC GTC GGC CGC TAC CGC A TGATCA T CGCCGGC GACGTC

2.OmpA (63mer)

GAATTC GCGGCCGC T TCTAG ATG AAA AAA ACC GCG ATT GCG ATT GCG GTG GCG CTG GCG GGC TTT GCG ACC GTG GCG CAG GCG T ACTAGT A GCGGCCG CTGCAG

CTTAAG CGCCGGCG A AGATC TAC TTT TTT TGG CGC TAA CGC TAA CGC CAC CGC GAC CGC CCG AAA CGC TGG CAC CGC GTC CGC A TGATCA T CGCCGGC GACGTC

3.PhoA (63mer) GAATTC GCGGCCGC T TCTAG ATG AAA CAG AGC ACC ATT GCG CTG GCG CTG CTG CCG CTG CTG TTT ACC CCG GTG ACC AAA GCG T ACTAGT A GCGGCCG CTGCAG

CTTAAG CGCCGGCG A AGATC TAC TTT GTC TCG TGG TAA CGC GAC CGC GAC GAC GGC GAC GAC AAA TGG GGC CAC TGG TTT CGC A TGATCA T CGCCGGC GACGTC

The yellow, green, blue and red part in pictures are primers we order from Biotech. The combination of these four sequences will produce the sticky ends of XbaI and SpeI. We ligase the yellow and blue part with green and red one respectively. Finally, we linked these two parts with plasmid backbones treated with Xbal and Spel.

Moreover, we should notice the possibility of antisense considering the sticky ends of XbaI and SpeI will pair together. After all the ligation and transformation process, we should use colony PCR to pick up out correct colony. On PSB1C3, there are two sequence: V2F and VR, provide the primer binding site. Therefore, while choose V2F and VR to be primers, we could still see band on gel even there’s no insertion. Our experiment design can intelligently solve this problem. We use one of our signal sequences to replace VR sequence, so the cases of antisense and without insert will not have result during electrophoresis.

All signal sequences are cloned and ligated on pSB1C3 plasmid backbone, which can be applied broadly as biobricks. By doing so, we significantly lower the cost compared to directly synthesize the nucleic acid sequences. And it is suitable for shorter gene sequence. We think it worth referencing by other iGEM teams.

PKEK sequence

In gram-negative bacteria, different types of protein secretion systems have been described , and we decided to put PKEK sequence directly behind the signal sequence, because it has only 18 nucleic acids. PKEK (18mer)

CGC AAA GAA AAA TAA TAA