Introduction
Biology is different from other engineering fields due to a lack of modular nature of the structure and function, which means the expression of gene will change when transferred from one species to another. What synthetic biology is dedicated to is the modularization and standardization of functional gene, to make the expression of it predictable in different chassis organism. Our project has registered 19 basic parts and 56 composite parts, all qualified for standardized requirement of RFC[10]. We have also tested these parts. We hope that everyone can utilize our parts based on our research through registry and that the information for parts can further be completed so that more people can make use of them.
Basic parts
| Part | Number | Part Name | Type | length |
|---|---|---|---|---|
| > | BBa_K3711019 | Panb1 | Regulatory | 399 |
| > | BBa_K3711001 | Pynr071c | Regulatory | 1188 |
| > | BBa_K3711015 | AOX1 | Regulatory | 937 |
| > | BBa_K3711017 | AOX1 terminator | Terminator | 247 |
| > | BBa_K3711002 | ROX1 | Coding | 1251 |
| > | BBa_K3711003 | FMO | Coding | 1395 |
| > | BBa_K3711004 | Laccase | Coding | 1602 |
| > | BBa_K3711005 | crtE | Coding | 927 |
| > | BBa_K3711006 | crtB | Coding | 948 |
| > | BBa_K3711007 | crtI | Coding | 1497 |
| > | BBa_K3711008 | LOX2 | Coding | 2709 |
| BBa_K3711009 | 4CL | Coding | 1704 | |
| BBa_K3711010 | ACC | Coding | 1800 | |
| BBa_K3711011 | CUS | Coding | 1227 | |
| BBa_K3711012 | curA | Coding | 1056 | |
| BBa_K3711013 | PepACS | Coding | 168 | |
| BBa_K3711014 | DsbC | Coding | 903 | |
| BBa_K3711016 | α-factor | Coding | 267 | |
| BBa_K3711018 | FMO dimer | Coding | 2769 |
Improvement
Flaving Containing-Monooxygenase (FMO) is an enzyme that can be used to produce indigo. This basic part has been registered by iGEM13_Berkeley as BBa_K1131000. We improved its function and registered as a new part BBa_K3711018.
Firstly, we optimized the codon of this gene according to the codon preference of P.pastoris to improve its expression level.
Secondly, research shows that FAD is the prosthetic group of FMO, NADPH is the cofactor of FMO. FMO-FAD-NADPH complex usually forms a asymmetric dimer as a unit to work. The enzyme-substrate complex formed when enzyme acts on substrate, has two dimers per unit[1].
So, we wanted to construct the two FMO fusion proteins named FMO dimer and expressed it, hoping that they could form a dimer more easily and improve their activity.
Then, we predicted the 3D structure of FMO dimer. The result shows that the structure could be formed and the dimer could combinate with substrate.
Therefore, we started to build FMO dimer and achieved success in expression. More details please click here.
Composite parts
| Part | Number | Part Name | Type | length |
|---|---|---|---|---|
| BBa_K3711039 | AOX1-α factor-ROX1-AOX1 Terminator | Composite | 2722 | |
| BBa_K3711040 | AOX1-α factor-FMO-AOX1 Terminator | Composite | 2866 | |
| BBa_K3711041 | AOX1-α factor-Laccase-AOX1 Terminator | Composite | 3073 | |
| BBa_K3711042 | AOX1-α factor-crtE-AOX1 Terminator | Composite | 2398 | |
| BBa_K3711043 | AOX1-α factor-crtB-AOX1 Terminator | Composite | 2419 | |
| BBa_K3711044 | AOX1-α factor-crtI-AOX1 Terminator | Composite | 2968 | |
| BBa_K3711045 | AOX1-α factor-LOX2-AOX1 Terminator | Composite | 4180 | |
| BBa_K3711046 | AOX1-α factor-4CL-AOX1 Terminator | Composite | 3175 | |
| BBa_K3711047 | AOX1-α factor-ACC-AOX1 Terminator | Composite | 3271 | |
| BBa_K3711048 | AOX1-α factor-CUS-AOX1 Terminator | Composite | 2698 | |
| BBa_K3711049 | AOX1-α factor-curA-AOX1 Terminator | Composite | 2527 | |
| BBa_K3711050 | AOX1-α factor-PepACS-AOX1 Terminator | Composite | 1639 | |
| BBa_K3711051 | AOX1-α factor-DsbC-AOX1 Terminator | Composite | 2374 | |
| BBa_K3711052 | Pynr071c-α factor-FMO dimer-AOX1 Terminator | Composite | 4491 | |
| BBa_K3711053 | Pynr071c-α factor-ROX1-AOX1 Terminator | Composite | 2973 | |
| BBa_K3711054 | Panb1-α factor-FMO-AOX1 Terminator | Composite | 2328 | |
| BBa_K3711055 | Pynr071c-α factor-Laccase-AOX1 Terminator | Composite | 3324 | |
| BBa_K3711056 | Panb1-α factor-crtE-AOX1 Terminator | Composite | 1860 | |
| BBa_K3711057 | Panb1-α factor-crtB-AOX1 Terminator | Composite | 1881 | |
| BBa_K3711058 | Panb1-α factor-crtI-AOX1 Terminator | Composite | 2430 | |
| BBa_K3711060 | Pynr071c-α factor-LOX2-AOX1 Terminator | Composite | 4431 | |
| BBa_K3711061 | Panb1-α factor-4CL-AOX1 Terminator | Composite | 2637 | |
| BBa_K3711062 | Panb1-α factor-ACC-AOX1 Terminator | Composite | 2733 | |
| BBa_K3711063 | Panb1-α factor-CUS-AOX1 Terminator | Composite | 2160 | |
| BBa_K3711064 | Pynr071c-α factor-curA-AOX1 Terminator | Composite | 2778 | |
| BBa_K3711065 | Panb1-α factor-PepACS-AOX1 Terminator | Composite | 1101 | |
| BBa_K3711066 | Pynr071c-α factor-DsbC-AOX1 Terminator | Composite | 2625 | |
| BBa_K3711067 | Pynr071c-ROX1-AOX1 Terminator | Composite | 2700 | |
| BBa_K3711068 | Panb1-FMO-AOX1 Terminator | Composite | 2055 | |
| BBa_K3711069 | Pynr071c-Laccase-AOX1 Terminator | Composite | 3051 | |
| BBa_K3711070 | Panb1-crtE-AOX1 Terminator | Composite | 1587 | |
| BBa_K3711071 | Panb1-crtB-AOX1 Terminator | Composite | 1608 | |
| BBa_K3711072 | Panb1-crtI-AOX1 Terminator | Composite | 2157 | |
| BBa_K3711073 | Pynr071c-LOX2-AOX1 Terminator | Composite | 4158 | |
| BBa_K3711074 | Panb1-4CL-AOX1 Terminator | Composite | 2364 | |
| BBa_K3711075 | Panb1-ACC-AOX1 Terminator | Composite | 2460 | |
| BBa_K3711076 | Panb1-CUS-AOX1 Terminator | Composite | 1887 | |
| BBa_K3711077 | Pynr071c-curA-AOX1 Terminator | Composite | 2505 | |
| BBa_K3711078 | Panb1-PepACS-AOX1 Terminator | Composite | 828 | |
| BBa_K3711079 | Pynr071c-DsbC-AOX1 Terminator | Composite | 2352 | |
| BBa_K3711080 | AOX1-α factor-Lycopene-AOX1 Terminator | Composite | 7801 | |
| BBa_K3711081 | AOX1-α factor-Curcumin-AOX1 Terminator | Composite | 9160 | |
| BBa_K3711082 | Panb1-α factor-Curcumin-AOX1 Terminator | Composite | 7546 | |
| BBa_K3711083 | Panb1-α factor-Lycopene-AOX1 Terminator | Composite | 6187 | |
| BBa_K3711084 | Panb1-Curcumin-AOX1 Terminator | Composite | 6727 | |
| BBa_K3711085 | Panb1-Lycopene-AOX1 Terminator | Composite | 5368 | |
| BBa_K3711086 | The synthesis and decolorization system of indigo | Composite | 8368 | |
| BBa_K3711087 | The synthesis and decolorization system of indigo | Composite | 7822 | |
| BBa_K3711088 | The synthesis and decolorization system of indigo | Composite | 14948 | |
| BBa_K3711089 | The synthesis and decolorization system of lycopene without signal peptide | Composite | 12242 | |
| BBa_K3711090 | The synthesis and decolorization system of lycopene without signal peptide | Composite | 13040 | |
| BBa_K3711091 | The synthesis and decolorization system of curcumin without signal peptide | Composite | 11948 | |
| BBa_K3711092 | The perm and straightening system | Composite | 6442 | |
| BBa_K3711093 | The perm and straightening system without signal peptide | Composite | 5896 |
How ingenious! It seems that constructing composite parts and full pathway takes engineering wisdom.