Design

1. Two plasmids by Daniel Ducat.

Figure 1. The structure of MF (p233) and PAM (p0015)

2. HydA was amplified from PAM using primers:
F_HydA_DD
5’ CAACCTCAAGATCGATATGGGGTTTTCCCAGTCACGACG 3’

Figure 2. Forward primer for amplification of HydA

R_HydA_DD
5’ CTCGGTACCCGGGGATCCTGTGGAATTGTGAGCGGATAACAATT 3’

Figure 3. Reverse primer for amplification of HydA

Figure 4. Amplification of HydA from PAM (p0015)

3. psba1 promoter was amplified from MF:
F_psbaI_DD
5’ GTTTATTTAACTAGTAGCGG 3’

Figure 5. Forward primer for amplification of psba1

R_psbaI_DD
5’CATATCGATCTTGAGGTTGTAAAGGGCAAGAGTC 3’

Figure 6. Reverse primer for amplification of psba1

Figure 7. Amplification of psba1 from MF (p233)

4. psba1+HydA insert was created using Polymerase Cycling Assembly (PCA):

Figure 8. Assembly of insert (psba1+HydA)

5. Then insert (psba1+HydA) was cut with BamHI-HF:

Figure 9. Digestion of insert with BamHI-HF

6. MF was digested using XhoI and BamHI-HF:

Figure 10. Digestion of MF (p233) with XhoI and BamHI-HF

7. Two pieces were ligated on BamHI-HF sticky ends and the other end was ligated using overlapping sequences:

Figure 11. Assembly of construct (p233+insert)

Team:NU Kazakhstan/Design

Design