Difference between revisions of "Part:BBa K2230004"
| (3 intermediate revisions by the same user not shown) | |||
| Line 4: | Line 4: | ||
| − | [[File:Mingdaophil1025-7.jpeg| | + | [[File:Mingdaophil1025-7.jpeg|600px]] |
| Line 18: | Line 18: | ||
| − | Erythromycin is a common selection marker for engineering lactic acid | + | Erythromycin is a common selection marker for engineering lactic acid bacteria. And teams [http://2013.igem.org/Team:Uppsala Uppsala] in 2013 and [http://2016.igem.org/Team:TMMU_China TMMU_China] in 2016 were using it to select the engineered Lactobacillus with erythromycin resistant strains. |
=== Cloning === | === Cloning === | ||
| − | RBS-EmR which was cut and gel-eluted from RBS-EmR/pSB1C3 (https://parts.igem.org/Part:BBa_K2230002 BBa_K2230002]) was assembled with CP29-RBS-aeBlue/pLBA169 ([https://parts.igem.org/Part:BBa_K2230000 BBa_K2230000]) | + | RBS-EmR which was cut and gel-eluted from RBS-EmR/pSB1C3 ([https://parts.igem.org/Part:BBa_K2230002 BBa_K2230002]) was assembled with CP29-RBS-aeBlue/pLBA169 ([https://parts.igem.org/Part:BBa_K2230000 BBa_K2230000]) |
Latest revision as of 06:42, 25 October 2017
RBS-EmR-CP29-RBS-aeBlue/pLBA169
The vector can be used to transform Lactobacillus acidophilus by chromosomal homologous recombination at the downstream location of slpA, which encodes a surface layer protein A. EmR (erythromycin resistance gene), which is driven by the high constitutive promoter of slpA, acts as a selection marker. And aeBlue gene, which is driven by the wide host range and high constitutive promoter CP29, acts as a reporter. The transformed L. acidophilus will be erythromycin resistant and express blue color proteins.
Design
To engineer Lactobacillus acidophilus by chromosome integration through homologous recombination, the selection of integration site is very important for successful recombination and not disturbing bacterial internal metabolism. Based on the method created by Grace L. Douglas and Todd R. Klaenhammer (Appl Environ Microbiol. 2011), the region between slpA gene (LBA0169) stop codon and the terminator was chosen as the intergenic insertion location. The gene of slpA encodes a surface-layer protein with a strong constitutive promoter activity, which can also drive the expression of the inserted gene.
Erythromycin is a common selection marker for engineering lactic acid bacteria. And teams [http://2013.igem.org/Team:Uppsala Uppsala] in 2013 and [http://2016.igem.org/Team:TMMU_China TMMU_China] in 2016 were using it to select the engineered Lactobacillus with erythromycin resistant strains.
Cloning
RBS-EmR which was cut and gel-eluted from RBS-EmR/pSB1C3 (BBa_K2230002) was assembled with CP29-RBS-aeBlue/pLBA169 (BBa_K2230000)
Reference
1. Directed chromosomal integration and expression of the reporter gene gusA3 in Lactobacillus acidophilus NCFM. Appl Environ Microbiol. 2011;77(20):7365-71.
2. Genetic engineering techniques for lactic acid bacteria: construction of a stable shuttle vector and expression vector for β-glucuronidase. Biotechnol Lett. 2014;36(2):327-35.
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal prefix found in sequence at 4909
Illegal suffix found in sequence at 1554 - 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 4909
Illegal SpeI site found at 1555
Illegal PstI site found at 1569
Illegal NotI site found at 1562
Illegal NotI site found at 4915 - 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 4909
Illegal XhoI site found at 3195
Illegal XhoI site found at 4087 - 23INCOMPATIBLE WITH RFC[23]Illegal prefix found in sequence at 4909
Illegal suffix found in sequence at 1555 - 25INCOMPATIBLE WITH RFC[25]Illegal prefix found in sequence at 4909
Illegal XbaI site found at 4924
Illegal SpeI site found at 1555
Illegal PstI site found at 1569 - 1000COMPATIBLE WITH RFC[1000]