Composite

Part:BBa_K3747606:Design

Designed by: Sanne van Doorn   Group: iGEM21_Wageningen_UR   (2021-10-21)
Revision as of 21:38, 21 October 2021 by Sanvdoorn (Talk | contribs) (→‎In-depth design)


Denitrification BAC


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 1348
    Illegal EcoRI site found at 1957
    Illegal EcoRI site found at 3310
    Illegal EcoRI site found at 16935
    Illegal EcoRI site found at 17859
    Illegal EcoRI site found at 24833
    Illegal SpeI site found at 3261
    Illegal PstI site found at 5615
    Illegal PstI site found at 5693
    Illegal PstI site found at 12267
    Illegal PstI site found at 13293
    Illegal PstI site found at 13539
    Illegal PstI site found at 14118
    Illegal PstI site found at 14805
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 1348
    Illegal EcoRI site found at 1957
    Illegal EcoRI site found at 3310
    Illegal EcoRI site found at 16935
    Illegal EcoRI site found at 17859
    Illegal EcoRI site found at 24833
    Illegal NheI site found at 7003
    Illegal NheI site found at 25343
    Illegal SpeI site found at 3261
    Illegal PstI site found at 5615
    Illegal PstI site found at 5693
    Illegal PstI site found at 12267
    Illegal PstI site found at 13293
    Illegal PstI site found at 13539
    Illegal PstI site found at 14118
    Illegal PstI site found at 14805
    Illegal NotI site found at 30477
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 1348
    Illegal EcoRI site found at 1957
    Illegal EcoRI site found at 3310
    Illegal EcoRI site found at 16935
    Illegal EcoRI site found at 17859
    Illegal EcoRI site found at 24833
    Illegal BglII site found at 813
    Illegal BglII site found at 2951
    Illegal BglII site found at 4947
    Illegal BglII site found at 5346
    Illegal BglII site found at 5763
    Illegal BglII site found at 7819
    Illegal BglII site found at 10451
    Illegal BamHI site found at 22426
    Illegal XhoI site found at 1529
    Illegal XhoI site found at 2632
    Illegal XhoI site found at 13020
    Illegal XhoI site found at 15918
    Illegal XhoI site found at 17157
    Illegal XhoI site found at 24137
    Illegal XhoI site found at 24902
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 1348
    Illegal EcoRI site found at 1957
    Illegal EcoRI site found at 3310
    Illegal EcoRI site found at 16935
    Illegal EcoRI site found at 17859
    Illegal EcoRI site found at 24833
    Illegal SpeI site found at 3261
    Illegal PstI site found at 5615
    Illegal PstI site found at 5693
    Illegal PstI site found at 12267
    Illegal PstI site found at 13293
    Illegal PstI site found at 13539
    Illegal PstI site found at 14118
    Illegal PstI site found at 14805
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 1348
    Illegal EcoRI site found at 1957
    Illegal EcoRI site found at 3310
    Illegal EcoRI site found at 16935
    Illegal EcoRI site found at 17859
    Illegal EcoRI site found at 24833
    Illegal SpeI site found at 3261
    Illegal PstI site found at 5615
    Illegal PstI site found at 5693
    Illegal PstI site found at 12267
    Illegal PstI site found at 13293
    Illegal PstI site found at 13539
    Illegal PstI site found at 14118
    Illegal PstI site found at 14805
    Illegal NgoMIV site found at 1285
    Illegal NgoMIV site found at 2306
    Illegal NgoMIV site found at 3076
    Illegal NgoMIV site found at 3923
    Illegal NgoMIV site found at 4225
    Illegal NgoMIV site found at 4715
    Illegal NgoMIV site found at 5252
    Illegal AgeI site found at 1768
    Illegal AgeI site found at 3772
    Illegal AgeI site found at 6314
    Illegal AgeI site found at 6860
    Illegal AgeI site found at 10390
    Illegal AgeI site found at 13430
    Illegal AgeI site found at 19220
  • 1000
    COMPATIBLE WITH RFC[1000]


Design Notes

Given that the denitrification machinery + antibiotic resistance is about 32kb, a BAC was designed. This was done in a way that it could be assembled in yeast. Therefore, a centromere (CEN), an autonomous replicating (ARS), and Leucine biosynthesis (LEU) were included in the design [1]. Leu was added because the yeast strain that would have been used is auxotrophic for leucine. Moreover, we used the PCC1fos backbonewhich is a low copy number plasmid. The rationale for this is that a large, high copy number plasmid >30kb can excessively burden bacteria and are prone to mutate [2].

Figure 1. Original plan workflow to study interaction between DT (BBa_K2607000) and HT (BBa_K2607001).

In-depth design

From 5' to 3', the 32 kb denitrification casette consists of:

1. lox66(BBa_K3747604), synthetic variant of loxP, added to facilitate Cre-Lox-mediated genomic insertion. Sequenced retrieved from [1].

2. Gentamycin resistance(BBa_K3910008), to select for successful transformation of the BAC

3. The periplasmic nitrate reductase (Nap) operon from P. stutzeri (BBa_K3747600) with at the 5’ end a ribosome binding site (RBS): (BBa_J34801).

Nap is the dissimilatory nitrate reductase, which converts nitrate (NO3-) into nitrite (NO2-). This is the first step of denitrification, and therefore added as the first denitrification enzyme in the BAC. The RBS is added to ensure transcription of the first gene of the operon, as during PCR amplification the native RBS could have been lost.

4. The nitrite reductase (Nir) operon from P. stutzeri(BBa_K3747601)with at the 5’ end a ribosome binding site (RBS):(BBa_J34801).

Nir is the nitrate reductase, which converts nitrite (NO2-) into nitric oxide (NO). Cytochromes and genes for heme d1 synthesis are present in the native operon. Without these co-factors, Nir would not have been active. Nir takes care of the second step of denitrification and was therefore added as the second enzyme in the BAC. The RBS is added to ensure transcription of the first gene of the operon, as during PCR amplification the native RBS could have been lost.

5. The nitric oxide reductase (Nor) operon from P. stutzeri(BBa_K3747602) with at the 5’ end a T7 promoter: BBa_K3633015,a RBS: (BBa_J34801).

Nor is the nitric oxide reductase, which converts nitric oxide (NO) into nitrous oxide (N2-O). Cytochromes and genes for heme d1 synthesis are present in the native operon. Without these co-factors, Nir would not have been active. Nor takes care of the second step of denitrification and was therefore added as the third enzyme in the BAC. The RBS is added to ensure transcription of the first gene of the operon, as during PCR amplification the native RBS could have been lost. ALthough the T7 phage's genome is 40kb long, The T7 polymerase is added to make sure the last two enzymes (Nor, Nos) are expressed.


6. The nitrous oxide reductase (Nos) operon from P. stutzeri(BBa_K3747603) with at the 5’ end a RBS: (BBa_J34801).

Nos is the nitrous oxide reductase, which converts into nitrous oxide (N2-O) into dinitrogen gas (N2). Nos takes care of the last step of denitrification and was therefore added as the last enzyme in the BAC. The RBS is added to ensure transcription of the first gene of the operon, as during PCR amplification the native RBS could have been lost.

7. lox71/2m(BBa_K3747605), synthetic variant of loxP, added to facilitate Cre-Lox-mediated genomic insertion. Sequence retrieved from [1].

Source

Pseudomonas stutzeri JM300

References

[1] N. Kouprina and V. Larionov, “Exploiting the yeast Saccharomyces cerevisiae for the study of the organization and evolution of complex genomes,” FEMS Microbiol. Rev., vol. 27, no. 5, pp. 629–649, Dec. 2003, doi: 10.1016/S0168-6445(03)00070-6. [2] G. DG, Y. L, C. RY, V. JC, H. CA, and S. HO, “Enzymatic assembly of DNA molecules up to several hundred kilobases,” Nat. Methods, vol. 6, no. 5, pp. 343–345, 2009, doi: 10.1038/NMETH.1318.