Team:Hong Kong HKU/Part Collection
Parts Overview
From the construct, our team wishes to maintain a specific phenotypic ratio of red and green fluorescence gene by going through flipping recombination event carried out by the action of Cre recombinase. To achieve this function, our team has managed to clone 2 constructs, one for the expression of phenotypic ratio, and the other one for the expression of CRE enzyme.
Construct for the expression of phenotypic ratio has the following parts:
- Lac Promoter
- RBS
- Lox site pairs in opposite direction
- GFP reporter gene
- Inverted RFP reporter gene
With different fluorescence gene in between the two Lox sites in opposite direction, the flipping recombination event will occur with the expression of CRE recombinase allowing only 1 fluorescence gene to be expressed at a time.
Construct for the expression of CRE recombinase has the following parts:
- pBAD promoter
- Weak/Strong RBS
- Mutagenized CRE recombinase system
pBAD promoter is used to less stress the host cell as it has low metabolic burden. Weak RBS was used in the construct which has high copy number whereas the strong RBS is used in low copy plasmids.
Future direction and construction of complex recombination system.
- Construction of a more complex recombination circuit with different recombinases
- Serine/Tyrosine recombinase
- All non-Cre recombinase parts can be viewed below
With combination of Serine and Tyrosine recombinases and different recombination sites, a more complex and conditional recombination circuit could be built for different needs.
| Part number | Basic/Composite | Part Type | Description | Base Pair Number |
|---|---|---|---|---|
| BBa_K3644004 | Basic | Protein Coding | WT cre (e coli codon optimized) | 1032 |
| BBa_K3644002 | Basic | Protein Coding | R32V (Mutant Cre) | 1032 |
| BBa_K3644001 | Basic | Protein Coding | R32M (Mutant Cre) | 1032 |
| BBa_K3644003 | Basic | Protein Coding | Y324F (Mutant Cre) | 1032 |
| BBa_K3644005 | Basic | Protein Coding | 303GVSdup (Mutant Cre) | 1032 |
| BBa_K3644000 | Composite | Reporter | Reverse strong RBS RFP degradation Tag | 922 |
| BBa_K3644006 | Basic | DNA | Lox5171 (Forward) | 34 |
| BBa_K3644007 | Basic | DNA | Lox5171 (Reverse) | 34 |
| BBa_K3644008 | Basic | Protein Coding | Bacteria codon optimized RfxCas13d protein | 2944 |
| BBa_K3644009 | Basic | Protein Coding | Bacteria codon optimized Bxb1 excisionase | 780 |
| BBa_K3644010 | Basic | Protein Coding | Human codon optimized Bxb1 excisionase | 807 |
| BBa_K3644011 | Basic | Protein Coding | Human codon optimized Bxb1 integrase | 1530 |
| BBa_K3644012 | Basic | Protein Coding | E coli codon optimised PhiC31 integrase | 1818 |
| BBa_K3644013 | Basic | Protein Coding | E coli codon optimised PhiC31 integrase Type II | 1848 |
| BBa_K36440014 | Basic | Protein Coding | Human codon optimised PhiC31 integrase | 1842 |
| BBa_K3644015 | Basic | Protein Coding | Human codon optimised PhiC31 integrase 2 fragments | 1875 |
| BBa_K3644016 | Basic | Protein Coding | E. coli codon optimized Hin integrase | 573 |
| BBa_K3644017 | Basic | Protein Coding | Original Hin integrase | 573 |
| BBa_K3644018 | Basic | Protein Coding | E. coli codon optimized Gin integrase | 582 |
| BBa_K3644019 | Basic | Protein Coding | Original Gin integrase | 582 |
| BBa_K3644020 | Basic | Protein Coding | E. coli codon optimized Fin invertase | 558 |
| BBa_K3644021 | Basic | Protein Coding | Fin invertase | 558 |