Team:Ionis Paris/Part Collection
Part Collection
To insert our genes of interest in our bacteria, we worked with the plasmid pSB1A3 (high copy number). It contains a resistance gene to ampicillin.
Composite Table
| Name | Type | Description | Length (bp) |
| BBa_K3933005 | Composite | Reporter system for Vitamin B12 using a riboswitch | 2041 |
| BBa_K3933006 | Composite | Lactate Dehydrogenase with an inducible Arabinose promoter | 2341 |
| BBa_K3933007 | Composite | Sensing system for Vitamin B12 inducing the production of lactate | 2433 |
| BBa_K3933011 | Composite | Optimised sensing system for Vitamin B12 inducing the production of lactate | 2435 |
| BBa_K3933013 | Composite | Ldh regulated by a Magnesium riboswitch | 2696 |
| BBa_K3933014 | Composite | Lactate dehydrogenase regulated by a Manganese riboswitch | 1429 |
| BBa_K3933015 | Composite | Lactate dehydrogenase regulated by a Fluoride riboswitch | 1330 |
| BBa_K3933016 | Composite | Lactate dehydrogenase regulated by miRNA 141 | 1456 |
| BBa_K3933017 | Composite | RFP regulated by miRNA 141 | 1142 |
| BBa_K3933018 | Composite | Standardized version of our sensor to be adapted for sequestering riboswitches | 2238 |
| BBa_K3933019 | Composite | Standardized version of our sensor to be adapted for riboswitches inducing the translation | 1363 |
BBa_K3933005
This part is a reporter system for Cobalamin (vitamin B12), using a riboswitch which acts as a sequester of the RBS located downstream of its sequence. It was inspired by a part created by Paris Bettencourt 2015 and modified by OUC Paris 2019 : BBa_K2904100 . This part is a composite part that can be divided into two units. The first unit is a Tet R protein (BBa_C0040) , which is regulated by a constitutive promoter (BBa_J23106) and a double terminator BBa_B0015 . However, the translation of the Tet R protein is also regulated by a Cobalamin Riboswitch (BBa_K3933003) , which sequesters the RBS (BBa_B0032) when bound to vitamin B12. The second unit is the reporter system related to the sensing of Vitamin B12 realised in the first part. It is composed of a Tet Operator (BBa_R0040) , which hinders the expression of the coding sequence placed after when it is bound by Tet R. It is followed by a mRFP (BBa_E1010) translated thanks to a RBS (BBa_B0032) . This unit is also closed by the double terminator BBa_B0015 . When the riboswitch isn't bound to Cobalamin, the RBS is not sequestered and Tet R is expressed, which hinders the expression of the RFP.
However, when Cobalamin (or vitamin B12) binds to the riboswitch, it sequesters the RBS, and the Tet R is not translated. With no Tet R binding the Tet Operator, RFP can be translated and quantified. Thus, the RFP can be detected when Cobalamin binds to the riboswitch, thanks to a double inhibition system.
BBa_K3933007
This part is a composite part that can be divided into two units. The first unit is a Tet R protein (BBa_C0040) , which is regulated by a constitutive promoter (BBa_J23106) and a double terminator BBA_B0015 . However, the translation of the Tet R protein is also regulated by a Cobalamin Riboswitch ( BBa_K3933003 ) , which sequesters the RBS (BBa_B0032) when bound to vitamin B12. The second unit is the reporter system related to the sensing of Vitamin B12 realised in the first part. It is composed of a Tet Operator (BBa_R0040) , which hinders the expression of the coding sequence placed after when it is bound by Tet R. It is followed by a Lactate Dehydrogenase (BBa_K3102006) translated thanks to a RBS (BBa_B0032) . This unit is also closed by the double terminator BBa_B0015 . When the riboswitch isn't bound to Cobalamin, the RBS is not sequestered and Tet R is expressed, which hinders the expression of the Lactate Dehydrogenase.
However, when Cobalamin (or vitamin B12) binds to the riboswitch, it sequesters the RBS, and the Tet R is not translated. With no Tet R binding the Tet Operator, RFP can be translated and quantified. Thus, the RFP can be detected when Cobalamin binds to the riboswitch, thanks to a double inhibition system.
Details and explanations on the parts BBa_K3933013 to BBa_K3933019 can be found on the Human Practice page .
RBS Table
| Name | Type | Description | Length (bp) |
| BBa_B0032 | Basic, RBS | Medium Ribosome binding site | 13 |
| BBa_K3933008 | Basic, RBS | RBS optimized for TetR by Salis Lab | 48 |
| BBa_K3933009 | Basic, RBS | RBS optimized for the Ldh by Salis Lab | 50 |
Spacer Table
| Name | Type | Description | Length (bp) |
| BBa_K3933001 | Basic, DNA | RBS spacer | 7 |
| BBa_K3933002 | Basic, DNA | RBS spacer | 6 |
| BBa_K3933004 | Basic, DNA | Terminator spacer | 20 |
Promoter Table
Name
Type
Description
Length (bp)
BBa_BI0500
Basic, Promoter
Arabinose promoter
1210
BBa_J23106
Basic, Promoter
Constitutive promoter
35
BBa_R0040
Basic, Promoter
Tet inducible promoter
54
Coding regions
Name
Type
Description
Length (bp)
BBa_E1010
Basic, coding region
Red Fluorescent Protein
706
BBa_C0040
Basic, coding region
Tet repressor protein
685
BBa_K3933004
Basic, coding region
Lactate dehydrogenase
1154
Other Basics
Name
Type
Description
Length (bp)
BBa_K3933003
Basic, regulatory
Cobalamin riboswitch
211
BBa_B0015
Basic, Terminator
Double terminator
129
BBa_K3933010
Basic, Scar
Zone of insertion of the riboswitch
129
BBa_K3933020
Basic, Other
Golden Gate Esp3I Cloning Site
91
BBa_K3933003
This part is a Vitamin B12 Riboswitch, which acts as a sequester of the RBS located downstream of the RNA sequence of the riboswitch. This riboswitch inhibits the translation of the coding sequence placed after, by hindering the binding of the ribosome to the RNA sequence. This riboswitch was found in Propinobacterium shermanii, upstream of the gene cbiB, which is a Cobalamin biosynthesis gene. It has been demonstrated that it is sensible to Cobalamin.
When searching for a riboswitch sequence to use for our project, we found this sequence which was previously used by team Paris Bettencourt 2015 and team OUC China 2019 ( BBa_K2904100 ) . However, this part was not referenced alone as a basic part, but already combined with a diverse components such as a lac promoter and an mRFP. Paris Bettencourt used the riboswitch as a Cobalamin biosensor, by coupling it directly first with a GFP, then with an mRFP. As their results were not optimal, team OUC China decided to add a stabilizer and a tuner to their construction to have better results. All these information can be found on the BBa_K2904100 page.
| Name | Type | Description | Length (bp) |
| BBa_BI0500 | Basic, Promoter | Arabinose promoter | 1210 |
| BBa_J23106 | Basic, Promoter | Constitutive promoter | 35 |
| BBa_R0040 | Basic, Promoter | Tet inducible promoter | 54 |
| Name | Type | Description | Length (bp) |
| BBa_E1010 | Basic, coding region | Red Fluorescent Protein | 706 |
| BBa_C0040 | Basic, coding region | Tet repressor protein | 685 |
| BBa_K3933004 | Basic, coding region | Lactate dehydrogenase | 1154 |
Other Basics
Name
Type
Description
Length (bp)
BBa_K3933003
Basic, regulatory
Cobalamin riboswitch
211
BBa_B0015
Basic, Terminator
Double terminator
129
BBa_K3933010
Basic, Scar
Zone of insertion of the riboswitch
129
BBa_K3933020
Basic, Other
Golden Gate Esp3I Cloning Site
91
BBa_K3933003
This part is a Vitamin B12 Riboswitch, which acts as a sequester of the RBS located downstream of the RNA sequence of the riboswitch. This riboswitch inhibits the translation of the coding sequence placed after, by hindering the binding of the ribosome to the RNA sequence. This riboswitch was found in Propinobacterium shermanii, upstream of the gene cbiB, which is a Cobalamin biosynthesis gene. It has been demonstrated that it is sensible to Cobalamin.
When searching for a riboswitch sequence to use for our project, we found this sequence which was previously used by team Paris Bettencourt 2015 and team OUC China 2019 ( BBa_K2904100 ) . However, this part was not referenced alone as a basic part, but already combined with a diverse components such as a lac promoter and an mRFP. Paris Bettencourt used the riboswitch as a Cobalamin biosensor, by coupling it directly first with a GFP, then with an mRFP. As their results were not optimal, team OUC China decided to add a stabilizer and a tuner to their construction to have better results. All these information can be found on the BBa_K2904100 page.
| Name | Type | Description | Length (bp) |
| BBa_K3933003 | Basic, regulatory | Cobalamin riboswitch | 211 |
| BBa_B0015 | Basic, Terminator | Double terminator | 129 |
| BBa_K3933010 | Basic, Scar | Zone of insertion of the riboswitch | 129 |
| BBa_K3933020 | Basic, Other | Golden Gate Esp3I Cloning Site | 91 |
This part is a Vitamin B12 Riboswitch, which acts as a sequester of the RBS located downstream of the RNA sequence of the riboswitch. This riboswitch inhibits the translation of the coding sequence placed after, by hindering the binding of the ribosome to the RNA sequence. This riboswitch was found in Propinobacterium shermanii, upstream of the gene cbiB, which is a Cobalamin biosynthesis gene. It has been demonstrated that it is sensible to Cobalamin.
When searching for a riboswitch sequence to use for our project, we found this sequence which was previously used by team Paris Bettencourt 2015 and team OUC China 2019 ( BBa_K2904100 ) . However, this part was not referenced alone as a basic part, but already combined with a diverse components such as a lac promoter and an mRFP. Paris Bettencourt used the riboswitch as a Cobalamin biosensor, by coupling it directly first with a GFP, then with an mRFP. As their results were not optimal, team OUC China decided to add a stabilizer and a tuner to their construction to have better results. All these information can be found on the BBa_K2904100 page.