Design
Design
![](https://static.igem.org/mediawiki/2019/c/ce/T--NU_Kazakhstan--Design.jpg)
1. Two plasmids by Daniel Ducat.
![](https://static.igem.org/mediawiki/2019/d/db/T--NU_Kazakhstan--designMFandPAM.jpg)
2. HydA was amplified from PAM using primers:
F_HydA_DD
5’ CAACCTCAAGATCGATATGGGGTTTTCCCAGTCACGACG 3’
![](https://static.igem.org/mediawiki/2019/f/fb/T--NU_Kazakhstan--HydA_forward_primer.jpg)
R_HydA_DD
5’ CTCGGTACCCGGGGATCCTGTGGAATTGTGAGCGGATAACAATT 3’
![](https://static.igem.org/mediawiki/2019/b/b0/T--NU_Kazakhstan--HydA_reverse_primer.jpg)
![](https://static.igem.org/mediawiki/2019/7/77/T--NU_Kazakhstan--Amplification_of_HydA.jpg)
3. psba1 promoter was amplified from MF:
F_psbaI_DD
5’ GTTTATTTAACTAGTAGCGG 3’
![](https://static.igem.org/mediawiki/2019/5/5e/T--NU_Kazakhstan--psbA1_forward_primer1.jpg)
R_psbaI_DD
5’CATATCGATCTTGAGGTTGTAAAGGGCAAGAGTC 3’
![](https://static.igem.org/mediawiki/2019/3/39/T--NU_Kazakhstan--psba1_forward_primer.jpg)
![](https://static.igem.org/mediawiki/2019/5/56/T--NU_Kazakhstan--Amplification_of_psbA1.jpg)
4. psba1+HydA insert was created using Polymerase Cycling Assembly (PCA):
![](https://static.igem.org/mediawiki/2019/2/21/T--NU_Kazakhstan--designassemblyinsert.jpg)
5. Then insert (psba1+HydA) was cut with BamHI-HF:
![](https://static.igem.org/mediawiki/2019/f/fd/T--NU_Kazakhstan--designdigestioninsert.jpg)
6. MF was digested using XhoI and BamHI-HF:
![](https://static.igem.org/mediawiki/2019/d/d5/T--NU_Kazakhstan--Digestion_of_MF%28p233%29_with_BamHI_and_XhoI.jpg)
7. Two pieces were ligated on BamHI-HF sticky ends and the other end was ligated using overlapping sequences:
![](https://static.igem.org/mediawiki/2019/b/b5/T--NU_Kazakhstan--designassemblyconstruct.jpg)