Parts
Table
Here is an overview of all the new parts that were used in our project. Altogether, more parts were designed for this project but not characterised well enough to contribute to the parts. Nevertheless, these constructs helped us to get an idea of how our sensing system works.
Nitric oxide sensing parts:
For the success of our project, we needed to build a nitric oxide sensor which could recognise lower nitric oxide concentrations and elicit a higher response to induction than previously described parts.
| Name | Type | Description | Designers | Length in bp | Picture for composite parts |
|---|---|---|---|---|---|
| BBa_K4387000 | Promoter | pNorVβ, nitric oxide inducible | Jana Mehdy | 256 | |
| BBa_K4387001 | codon-optimized NorR | coding for transcription factor NorR | Jana Mehdy, Caua Westmann | 1515 | |
| BBa_K4387003 | piGEM1 (negative control) | coding for sfGFP and NorR, does not contain any promoter | Jana Mehdy, Lea Brüllmann, Marine Mausy | 2410 |
|
| BBa_K4387004 | piGEM3 | coding for sfGFP and NorR under the control of inducible ETH promoter pNorV | Jana Mehdy, Lea Brüllmann, Marine Mausy | 2713 |
|
| BBa_K4387005 | piGEM2_1RBS | coding for sfGFP and NorR under the control of inducible pNorVβ. This construct contains 1 RBS in front of sfGFP | Jana Mehdy, Lea Brüllmann, Marine Mausy | 2674 |
|
| BBa_K4387006 | piGEM2_2RBS | coding for sfGFP and NorR under the control of inducible pNorVβ. This construct contains 2 RBSs in front of sfGFP | Jana Mehdy, Lea Brüllmann, Marine Mausy | 2697 |
|
| BBa_K4387007 | piGEM2_3RBS | coding for sfGFP and NorR under the control of inducible pNorVβ. This construct contains 3 RBSs in front of sfGFP | Jana Mehdy, Lea Brüllmann, Marine Mausy | 2717 |
|
| BBa_K4387009 | piGEM2_2RBS without a positive feed-back loop | coding for sfGFP under the control of inducible pNorVβ. This construct contains 2 RBSs in front of sfGFP | Jana Mehdy, Lea Brüllmann, Marine Mausy | 1156 |
|
| BBa_K4387020 | RBS | strong RBS | Jana Mehdy, Marine Mausy | 12 |
Nanobody constructs:
To dampen inflammation, we needed a construct that could interfer in the pro-inflammatory signaling. To fulfill this mission, we decided to use a nanobody against the cytokine TNFα. First, several nanobody candidates were tested and characterized to find the best suitable construct.
| Name | Type | Description | Designers | Length in bp | Picture for composite parts |
|---|---|---|---|---|---|
| BBa_K4387996 | VHH#2B | Monovalent Anti-Tumour Necrosis Factor Nanobody (VHH#2B) | Nathalie Weibel | 369 | |
| BBa_K4387995 | VHH#3E | Monovalent Anti-Tumour Necrosis Factor Nanobody (VHH#3E) | Nathalie Weibel | 387 | |
| BBa_K4387994 | VHH#12B | Monovalent Anti-Tumour Necrosis Factor Nanobody (VHH#12B) | Nathalie Weibel | 369 | |
| BBa_K4387993 | Biv. VHH#2B | Bivalent Anti-Tumour Necrosis Factor Nanobody (biv. VHH#2B) | Nathalie Weibel | 774 | |
| BBa_K4387992 | Biv. VHH#3E | Bivalent Anti-Tumour Necrosis Factor Nanobody (biv. VHH#3E) | Nathalie Weibel | 810 | |
| BBa_K4387991 | Biv. VHH#12B | Bivalent Anti-Tumour Necrosis Factor Nanobody (biv. VHH#12B) | Nathalie Weibel | 774 | |
| BBa_K4387990 | VHH#3E + VHH#2B | Bivalent Anti-Tumour Necrosis Factor Nanobody (VHH#3E + VHH#2B) | Nathalie Weibel | 792 | |
| BBa_K4387989 | VHH#2B + VHH#12B | Bivalent Anti-Tumour Necrosis Factor Nanobody (VHH#2B + VHH#12B) | Nathalie Weibel | 774 | |
| BBa_K4387988 | VHH#3E + VHH#12B | Bivalent Anti-Tumour Necrosis Factor Nanobody (VHH#3E + VHH#12B) | Nathalie Weibel | 792 |
Secretion system:
To allow the secretion of the produced nanobodies into the extracellular space, we decided to make use of the hemolysin A secretion system. Since TolC is already endogenously expressed in many E. coli strains, we only needed to encode the other two subunits (HlyB and HlyD) and mark our nanobody with the hemolysin A secretion tag.
| Name | Type | Description | Designers | Length in bp | Picture for composite parts |
|---|---|---|---|---|---|
| BBa_K4387999 | HlyB | HlyB of the hemolysin A one-step secretion system | Nathalie Weibel | 2124 | |
| BBa_K4387998 | HlyD | HlyD of the hemolysin A one-step secretion system | Nathalie Weibel | 1437 | |
| BBa_K4387997 | HlyA-tag | HlyA-tag obtained from the hemolysin A one-step secretion system | Nathalie Weibel | 654 | |
| BBa_K4387987 | Secretion system | Hemolysin A secretion system for E. coli | Nathalie Weibel | 3734 |
|
| BBa_K4387986 | Monovalent nanobody expression | Monovalent nanobody expression with the HlyA secretion signal (VHH#2B) | Nathalie Weibel | 2421 |
|
| BBa_K4387985 | Monovalent nanobody expression | Monovalent nanobody expression with the HlyA secretion signal (VHH#3E) | Nathalie Weibel | 2439 |
|
| BBa_K4387984 | Monovalent nanobody expression | Monovalent nanobody expression with the HlyA secretion signal (VHH#12B) | Nathalie Weibel | 2421 |
|
| BBa_K4387983 | Bivalent nanobody expression | Bivalent nanobody expression with the HlyA secretion signal (biv. VHH#2B) | Nathalie Weibel | 2826 |
|
| BBa_K4387982 | Bivalent nanobody expression | Bivalent nanobody expression with the HlyA secretion signal (biv. VHH#3E) | Nathalie Weibel | 2862 |
|
| BBa_K4387981 | Bivalent nanobody expression | Bivalent nanobody expression with the HlyA secretion signal (VHH#3E + VHH#2B) | Nathalie Weibel | 2844 |
|
| BBa_K4387980 | Bivalent nanobody expression | Bivalent nanobody expression with the HlyA secretion signal (VHH#2B + VHH#12B) | Nathalie Weibel | 2826 |
|
| BBa_K4387979 | Bivalent nanobody expression | Bivalent nanobody expression with the HlyA secretion signal (VHH#3E + VHH#12B) | Nathalie Weibel | 2844 |
|
Final construct:
Once all systems have been established and the best suited constructs identified, we Gibson-assembled a final plasmid, which would allow the inducible production of nanobodies upon nitric oxide sensing. The secretion system plasmid encoding for the secretion pore stayed the same. (See BBa_K4387987)
| Name | Type | Description | Designers | Length in bp | Picture for composite parts |
|---|---|---|---|---|---|
| BBa_K4387978 | Nitric Oxide Sensing Genetic Circuit | NO-induced monovalent nanobody expression | Nathalie Weibel | 3054 |
|