Team:Tongji China/Process

Tongji iGEM - Process

Process

Here is how our constructs were made.

Backbone

Clone pleP, Gal4, Gal80 and TH

Gal4 (2646bp) integrated with UAS-Gal4 system, and pleP (452bp) and Gal80ts (1311bp) are from the genome of Drosophila melanogaster integrated with a mutant Gal80ts. Because of the repetitive sequence on the TH, it is difficult to clone so we order the synthetic pUAST (double digestion with EcoRI and XbaI)-TH from GENEWIZ®.

Digest pUAST-3xHA

For constructing the plasmid pleP-Gal4 and pleP-Gal80ts, avoiding the influence of UAS on the backbone we should remove it. We remove UAS by BamHI digestion.

A) pUAST-3xHA
B) pUAST-3xHA BamHI (for 6)

Assembly

To assembly the digested backbone and fragment. We take two ways: to insert a new MCS into the backbone (done), then use a method of double digestion to insert two fragments one by one (failed), or to single digest, then use seamless cloning to insert two fragment together (success).

A) To insert a new MCS into the backbone (done), then use a method of double digestion to insert two fragments one by one (failed)
1. Transform backbone with a new MCS

2. BglII digestion to insert pleP (done)

Digestion
A) pUAST-oligo BglII
B) pUAST-oligo

pUAST-oligo-pleP

3. pUAST-new MCS-pleP digestion (done) to insert Gal4 and Gal80ts (failed)

Digestion
A) pUAST-new MCS -pleP
B) pUAST-new MCS -pleP NotI
C) pUAST-new MCS -pleP HpaI
D) pUAST-new MCS -pleP NotI&HpaI (For 11)

B) To single digest, then use seamless cloning to insert two fragment together (success)
1. BamHI digestion (2 done)
2. Seamless cloning to insert two fragment together (done)
We have done the enzyme digestion to verify them

pUAST-pleP-Gal4
A) pUAST-pleP-Gal4
B) pUAST-pleP-Gal4 PstI

pUAST-pleP-Gal80ts
A) pUAST-pleP-Gal80ts
B) pUAST-pleP-Gal80ts KpnI

pUAST-UAS-TH
A) pUAST-UAS-TH
B) pUAST-UAS-TH BglII

Ignis Fly

Tongji_China iGEM 2017 Team
Protocol