Recombination Device composed by Cre-lox71
Recombination Device composed by lox71-Cre recombinase
This device was created to express the Cre recombinase enzyme and a target gene under the control of the same promoter (promoter not included in this part). The target gene needs to be amplified by PCR using primers that will insert a loxP and a lox66 sites flanking the sequence in order to integrate it in the lox71 recombination site located upstream the Cre recombinase gene. Once this device is located under the control of the desired promoter, it will express the Cre recombinase that recognizes the lox sites and inserts the target gene under the control of the same promoter. The Cre expression levels are critical for the recombination, we created two different devices: A (Cre-Lox71) and B (Lox71-Cre) for testing if inserting the target gene either upstream or downstream the Cre gene will affect the expression efficiency of the target gene.
We used the lox71 submitted by our group, which correct the part BBa_I718017 and BBa_K537020 when compared with the published sequence at http://www.ncbi.nlm.nih.gov/pmc/articles/PMC137435/
We synthesized the lox71 site and then we assembled it with the Cre recombinase part BBa_J61047
Constitutive promoter+37℃ induced RBS+tetR+double terminator+Ptet+RBS+GFP+double terminator
We want to utilize a temperature-regulated mechanism to control the expression period of a crystal protein. Therefore, we took advantage of the interaction between 37?? C induced RBS, tetR gene and pTet promoter to create the temperature regulating circuit. The following is a description of our prescribed strategy. First, we use the constitutive promoter, 37?? C induced RBS (K115002), the tetR gene and terminators. We will call this part the ???Regulator???.
37℃ induced RBS + tetR + double terminator
It is a transcriptional unit including 37℃ induced RBS + tetR + double terminator. It can be added various promoter to adapt your conditions.
To regulate following genes by temperature. To maintain cells at 37?? C ↑ : the tetR protein be generated and repress Ptet's circuit. To release cells at 37?? C ↓ : the tetR protein not be generated and do not repress Ptet's circuit.
the 37℃ induced RBS cames from iGEM08_TUDelft
Constitutive promoter + 37℃ induced RBS + tetR + double terminator
we took advantage of the interaction between 37?? C induced RBS, tetR gene and pTet promoter to create the temperature regulating circuit. The following is a description of our prescribed strategy. First, we use the constitutive promoter, 37?? C induced RBS (K115002), the tetR gene and terminators. We will call this part the ???Regulator???. We speculate that when the ???Regulator??? and ???Production??? sections are combined, we can achieve a high bacteria growth rate and close to no target protein producing rate at T>37??C, and low bacteria growth rate and high target protein producing rate at T<37??C.
To regulate following gene by temperature
This device was created to express the Cre recombinase enzyme and a target gene under the control of the same promoter (promoter not included in this part). The target gene needs to be amplified by PCR, using primers that will insert a loxP and a lox66 sites flanking the sequence, in order to integrate it in the lox71 recombination site. For this part the lox71 is located downstream the Cre recombinase gene. An alternative construction is submitted in the part BBa_K886001. Once this device is located under the control of the desired promoter, it will express the Cre recombinase that recognizes the lox sites and inserts the target gene under the control of the same promoter. The Cre expression levels are critical for the recombination, we created two different devices: BBa_K886002 (Cre-Lox71) and BBa_K886001 (Lox71-Cre) for testing if inserting the target gene either upstream or downstream the Cre gene will affect the expression efficiency of the target gene
We used the lox71 submitted by our group BBa_K886000, which corrected the parts BBa_I718017 and BBa_K537020. Those parts are wrong when compared with the published sequence at http://www.ncbi.nlm.nih.gov/pmc/articles/PMC137435/
We synthesized the lox71 site, then we assembled it downstream the Cre recombinase gene(BBa_J61047).
This part is a differs from BBa_P1011 in that the mutations introduced in BBa_P1011 to remove restriction sites are not present in this part. It does have the "normal" ccd promoter and ccdB coding region. It also has a double TAA stop codon as per BioBrick conventions.
ccdA- was eliminated since it is an inactive form of ccdA and should not be required for the toxic phenotype of ccdB.
This part is derived from BBa_P1010 but it lacks ccdA-.