From 2008.igem.org
Construction of pRL1383a
- The BioBrick parts of pRL1383a are to be ligated in a series of experiments.
Parts
Units of pRL1383a Construct
| Name
| Status
| Quantity
| Individual construct
|
| OriV
| PCR amplified 7/30
| ?
| pSB1A3::OriV
|
| rep region
| PCR amplified 7/29
| ~960ng
| Plac-RBS-rep
|
| omega interposon
| PCR amplified 7/30
| ?
| pSB1A3::OriV
|
| aadA gene
| PCR amplified 7/29
| 128ng
| Plac-RBS-aadA-TT
|
| oriT
| Verified in vector 7/30
| 0(need plasmid prep, glycerol stock)
| pSB1A3::OriT
|
| Plac[http://partsregistry.org/Part:BBa_I14032 BBa_I14032]
| verified by colony PCR
| need glycerol stock and plasmid prep
| Plac-RBS-aadA-TT, Plac-RBS-rep
|
| RBS, efficiency 1.0[http://partsregistry.org/Part:BBa_B0034 BBa_B0034]
| plasmid available
| ? need to check
| Plac-RBS-aadA-TT, Plac-RBS-rep
|
| Terminator [http://partsregistry.org/Part:BBa_B0015 BBa_B0015]
| plasmid prep 7/30
| needs quantification
| Plac-RBS-aadA-TT
|
| BioBrick Base Vector[http://partsregistry.org/Part:BBa_I51020 BBa_I51020]
| plasmid prep 7/30
| needs quantification
| all parts will be inserted
|
Methods
- Round 1 of Restriction Digest and Ligation
Restriction Digest & Ligation: Round 1
| Name
| Digest 1
| Digest 2
| Ligation to
| Product
|
| mob
| XbaI & PstI
| n/a
| [http://partsregistry.org/Part:BBa_B0024 BBa_B0024]
| TT-mob
|
| rep
| XbaI & PstI
| n/a
| [http://partsregistry.org/Part:BBa_R0010 BBa_R0010]
| Plac-rep
|
| oriV
| XbaI & PstI
| n/a
| [http://partsregistry.org/Part:pSB1A3 pSB1A3]
| pSB1A3::oriV
|
| aadA region
| XbaI & PstI
| n/a
| [http://partsregistry.org/Part:BBa_R0010 BBa_R0010]
| Plac-aadA
|
| oriT
| XbaI & PstI
| n/a
| [http://partsregistry.org/Part:pSB1A3 pSB1A3]
| pSB1A3::oriT
|
| pSB1A3
| XbaI & PstI
| n/a
| see oriT and oriV
| see oriT and oriV
|
| [http://partsregistry.org/Part:BBa_B0024 BBa_B0024]
| SpeI
| PstI
| see mob region above
| see mob region above
|
| [http://partsregistry.org/Part:BBa_R0010 BBa_R0010]
| SpeI
| PstI
| see rep and aadA region above
| see rep and aadA region above
|
- In gel purification of ligation products
- Round 2 of Restriction Digest and Ligation
Restriction Digest & Ligation: Round 2
| Name
| Digest 1
| Digest 2
| Ligation to
| Product
|
| TT-mob
| EcoRI
| SpeI
| [http://partsregistry.org/Part:BBa_B0015 BBa_B0015]
| TT-mob-TT
|
| Plac-aadA
| EcoRI
| SpeI
| [http://partsregistry.org/Part:BBa_B0015 BBa_B0015]
| Plac-aadA -TT
|
| [http://partsregistry.org/Part:BBa_B0015 BBa_B0015]
| EcoRI
| XbaI
| see mob and aadA above
| see mob and aadA above
|
- Transformation
- Verification with Colony PCR
Results
Discussion
- What was learned and how to do future experiments differently.
[http://manoa.hawaii.edu/ 
][http://manoa.hawaii.edu/ovcrge/ 
][http://www.ctahr.hawaii.edu 
]
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