Team:HUST-China/Part collection
Protocol
Part1: Bacteria and culture medium
| Part Number | Name | Description |
|---|---|---|
| BBa_K2533000 | RBS-ldhD-RBS-lldP | ldhD can produce lactate, and lldP can release it from cytoplasm to the environment |
| BBa_K2533001 | RBS-ldhDC-RBS-lldP | ldhDC can produce lactate, lldP can release it from cytoplasm to the environment |
| BBa_K2533002 | RBS-ldhDnARSdR-RBS-lldP | ldhDnARSdR can produce lactate, and lldP can release it from cytoplasm to the environment. |
| BBa_K2533003 | RBS-ldhDARSdR-RBS-lldP | ldhDARSdR can produce lactate, and lldP can release it from cytoplasm to the environment. |
| BBa_K2533014 | RBS-TH-RBS-gldA-RBS-LLDP | produce and release lactate |
| BBa_K2533016 | RBS-ldhD-FLAG-lldP-his tag | ldhD can produce lactate, lldP can release it from cytoplasm to the environment, and tags will show the expression of the protein. |
| BBa_K2533017 | RBS-ldhDC-FLAG-lldP-his tag | ldhDC can produce lactate, lldP can release it from cytoplasm to the environment, and tags will show the expression of the protein. |
| BBa_K2533018 | RBS-ldhDnARSdR-FLAG-RBS-lldP-his tag | ldhDnARSdR can produce lactate, lldP can release it from cytoplasm to the environment, and tags will show the expression of the protein. |
| BBa_K2533019 | RBS-ldhDARSdR-FLAG-RBS-lldP-his tag | ldhDARSdR can produce lactate, lldP can release it from cytoplasm to the environment, and tags will show the expression of the protein. |
Circuit：
Figure 1. Circuit of ldhD-lldP.
Figure 2. Circuit of ldhDC-lldP.
Figure 3. Circuit of ldhDnARSdR-lldP.
Figure 4. Circuit of ldhDARSdR-lldP.
The the secondary circuit is the optimized type of the firist one,while the fourth circuit is the optimized type of the third one.Last three circuit is trying to improve the production of lactate.
Figure 6. modified circuit of ldhD-lldP.
The fifth circuit could help to contribute to produce more ldhD and lactate.
Figure 7. modified circuit of ldhDC-lldP.
Figure 8. modified circuit of ldhDnARSdR-lldP.
Figure 9. modified circuit of ldhDARSdR-lldP.
This four circuit is used to show the expression of protein.
Part2: Rhodopseudomonas palustris
| Part Number | Name | Description |
|---|---|---|
| BBa_K2533045 | RBS-mleS | It encodes malate dehydrogenase. |
| BBa_K2533046 | RBS-lldP | It encodes L-lactate permease. |
| BBa_K2533047 | RBS-ldhA | It encodes D-lactate dehydrogenase. |
| BBa_K2533048 | RBS-mleS-RBS-lldP | It encodes malate dehydrogenase & L-lactate permease. |
| BBa_K2533049 | RBS-lldP-RBS-ldhA | It encodes L-lactate permease & D-lactate dehydrogenase. |
| BBa_K2533050 | mleS-lldp-ldhA | It encodes malate dehydrogenase & L-lactate permease & D-lactate dehydrogenase. |
| BBa_K2533051 | mleS-lldP-TT | It encodes malate dehydrogenase & L-lactate permease. |
| BBa_K2533052 | RBS-lldP-RBS-ldhA-TT | Product and transport of lactic acid. |
| BBa_K2533053 | RBS-mleS-RBS-lldP-RBS-ldhA-TT | Product and transport of lactic acid. |
| BBa_K2533054 | RBS-ldhA-RBS-lldP | Product and transport of lactic acid. |
| BBa_K2533055 | RBS-ldhA-RBS-lldP-TT | Product and transport of lactic acid. |
mleS：malate dehydrogenase, the conversion of malic acid to L-lactate.
ldhA：fermentative D-lactate dehydrogenase, NAD-dependent, convert pyruvate to D-lactate.
lldP：L-lactate permease, the lactate is transported out of the cell.
To validate our modeling results, we continue to build three different gene circuits which can determine the highest lactate production efficiency of our total circuit:
Part3: Shewanella oneidensis
| Part Number | Name | Description |
|---|---|---|
| BBa_K2533030 | RBS-dld | It encodes D-lactate dehydrogenase. |
| BBa_K2533031 | RBS-lldEFG | produce L-lactate dehydrogenase complex protein. |
| BBa_K2533032 | RBS-dld-TT-Ptac-RBS-lldEFG | It encodes FAD-dependent D-lactate dehydrogenase and L-lactate dehydrogenase complex protein. |
| BBa_K2533033 | RBS-gapA | It encodes glyceraldehyde-3-phosphate dehydrogenase. |
| BBa_K2533034 | RBS-mdh | It encodes NAD dependent malate dehydrogenase. |
| BBa_K2533035 | RBS-gapA-RBS-mdh-TT | It encodes glyceraldehyde-3-phosphate dehydrogenase and NAD dependent malate dehydrogenase. |
| BBa_K2533036 | RBS-pflB | It encodes pyruvate formate-lyase. |
| BBa_K2533037 | RBS-fdh | It encodes formate dehydrogenase cytochrome b. |
| BBa_K2533038 | RBS-pflB-RBS-fdh | It encodes pyruvate formate-lyase and formate. |
| BBa_K2533039 | RBS-gapA-RBS-mdh-TT-Ptac-RBS-pflB-RBS-fdh | It encodes glyceraldehyde-3-phosphate dehydrogenase, NAD dependent malate dehydrogenase, pyruvate formate-lyase and formate dehydrogenase cytochrome b. |
This part collection well documents ten circuits in Shewanella oneidensis of our project with every detailed characterization of the transcriptional related proteins and promoters. The collection contains two primary circuits, where tac promoter and enhanced RBS are applied.
Circuit One:
This circuit aims at improving the efficiency of lactate generation. It contains two genes encoding D-lactate dehydrogenase and L-lactate dehydrogenase.
Circuit two:
The secondary circuit is try to improve the production of NADH as which includes four genes responsible for certain reaction in the metabolism system.
To better verify and quantify the functions of the circuits, the secondary circuits and single sectors of them are included, and as a result, not only can this part collection be applied to realize an optimized generation of electricity in Shewanella oneidensis, but also it offers comprehensive options for users to achieve certain functions with different compositions of these circuits.
