Part:BBa_K2172010
Tac Promoter-RBS-GST-Thrombin Protease-TEV-GFP-Terminator
This part is a gene circuit that can be used to test whether it is being expressed or not. It comprises of a ribosome binding site (RBS) for ribosomes to bind, a GST label for purification, a thrombin protease cleavage site plus a TEV protease cleavage site, a GFP detection unit, and a terminator. When transformed into bacteria, e.g. E. coli, it can be used to test whether the bacteria are expressing the part not. Other parts, such as the gene SmCPS1, can be inserted into this part via digestion on the thrombin site and the TEV site.
This circuit is responsible for the function of all sequence with a terminator waiting for the SmCPS1 protein to insert among Thrombin protease and TEV restriction cleavage sites. The above circuit without target gene SmCPS1 is constructed mainly to test whether it produces SmCPS1. If the circuit do work, it would result in the expression of the GFP gene and emission of green fluorescent light.
Name: BBa_K2172010
TAC-F primer: TCTAGATGACAATTAATCATCGGCT
TE-R primer: ACTAGTATGTATTTAGAAAAATAAACAAATAGG
Description: Promoter and RBS added, GST labeled, coding with Thrombin and TEV restriction enzyme cleavage sites, GFP detector, and Terminator.
Function: See BBa_K2172010 in part registry.
Length: 1719bp
We separated target gene SmCPS1 from the whole circuit adding GFP detector and terminator as terminal signal to test its function, which ensure the further protein secretion.
Green fluorescent light of E. coli under microscope. The E. coli transformed from white to fluorescent Green, because GFP segment inserted into plasmid result in unique detective color. We obtained the average expressive value of the green fluorescence in the biobrick part.
Tac Promoter (29bp) ---RBS (14bp) ---GST (757) ---Thrombin protease (18bp) ---SmCPS1 (2382bp) ---TEV (21bp) ---GFP (720bp) ---Terminator (160bp)
We constructed this complete circuit finally to express our gene SmCPS1 and secrete its protein from E. coli. The whole gene circuit consists of K2172010 part including SmCPS1. Thus we labeled GFP segment to test functions. As pure protein SmCPS1 have successfully been extracted, we are able to synthesize one process of the compounds production.
Tac Promoter (29bp):
Function: The tac promoter, a strong hybrid promoter, is used to control and increase the expression levels of a target gene and is used in the over-expression of recombinant proteins, produced from the combination of promoters from the trp and lac operons.
Sequence: tgacaattaatcatcggctcgtataatgt
RBS (14bp):
Function: A ribosome binding site (RBS) is a sequence of mRNA. It is used to lead the ribosome to the right position on the mRNA during the beginning of translation
Sequence:
tcacacaggaaaca
GST (757bp):
Function: By inserting the GST DNA coding sequence next to protein of interest, the GST can be added to a protein of interest to purify the fusion protein from solution in a process known as a pull-down assay. The strong binding affinity of GFP beads coated with the compound can be added to the protein mixture. As a result, the protein of interest attached to the GST will stick to the beads, isolating the protein from the rest of those in solution.
Sequence:
atgtcccctatactaggttattggaaaattaagggccttgtgcaacccactcgacttcttttggaatatcttgaagaaaaatatgaagagcatttgtatgagcgcgatgaaggtgataaatggcgaaacaaaaagtttgaattggg
tttggagtttcccaatcttccttattatattgatggtgatgttaaattaacacagtctatggccatcatacgttatatagctgacaagcacaacatgttgggtggttgtccaaaagagcgtgcagagatttcaatgcttgaaggagcg
gttttggatattagatacggtgtttcgagaattgcatatagtaaagactttgaaactctcaaagttgattttcttagcaagctacctgaaatgctgaaaatgttcgaagatcgtttatgtcataaaacatatttaaatggtgatcatgtaa
cccatcctgacttcatgttgtatgacgctcttgatgttgttttatacatggacccaatgtgcctggatgcgttcccaaaattagtttgttttaaaaaacgtattgaagctatcccacaaattgataagtacttgaaatccagcaagtata
tagcatggcctttgcagggctggcaagccacgtttggtggtggcgaccatcctccaaaatcggatctggttccgcgtggatccccgggaatttccggtggtggtggtggaattctagactccatgggtcgactcgagctcaag
cttattcatcgtgactgac
Thrombin protease (18bp):
Sequence: CTGGTTCCGCGTGGATCC
TEV (21bp):
Function: Thrombin protease, a DNA segment of pGEX-KG vector containing Bam Hl cloning site after GST, and TEV added at front of the GFP, are the two type of restriction enzyme cleavage sites inserted into tanshinone gene circuit in order to purify the SmCPS1 enzyme.
Sequence: GAAAACCTGTATTTTCAGGGC
GFP (720bp):
Function: The gene for the production of GFP is incorporated into the genome of the organism in the region of the DNA that codes for the target proteins and that is controlled by the same regulatory sequence; that is, the gene's regulatory sequence now controls the production of GFP, where expression of GFP can be used as a detection device for a particular characteristic.
Sequence:
ATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGC
GATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCA
GCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACC
CGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACT
ACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACT
ACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACAT
GGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGTAA
Terminator (160bp):
Function: Termination (T terminator) is the DNA sequence of the RNA polymerase termination signal of transcription, which is a structure at A structure in the poly (A) site downstream around 160bp
Sequence:
AGCGATAGCGGAGTGTATAATTCTTGAAGACGAAAGGGCCTCGTGATACGCCTATTTTTATAGGTTAATGTCATGATAATAATGGTTTCTTAGACGTCAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATT
SmCPS1 sequencing result (before Site-directed mutagenesis)： ATGGCCTCCTTATCCTCTACAATCCTCAGCCGCTCTCCGGCGGCCCGCCGCAGAATTACGCCGGCATCGGCTAAGCTTCACCTGCCGGAATGTTTCGCCATCAGTGCATG
GATGGGCAGCAGCACTAAAAACCTTTCTCTCAGCTACCAACTTAATCACAAGAAAATATCAGTTGCCACAGTAGATGCGCCGCAGGTGCATGACCACGACGGCACTACCGT
TCATCAAGGCCATGATGCGGTGAAGAATATTGAGGATCCCATTGAATACATCAGGACGTTGTTGAGGACGACGGGGGACGGGAGAATAAGCGTGTCGCCGTACGACACGG
CGTGGGTGGCGATGATCAAGGACGTGGAGGGGCGGGACGGGCCCCAGTTCCCCTCCAGCCTCGAGTGGATCGTGCAGAATCAACTCGAGGATGGATCGTGGGGCGATC
AGAAGCTTTTCTGCGTCTACGATCGCCTCGTCAATACCATCGCGTGCGTGGTAGCCTTGAGATCGTGGAATGTTCATGCTCACAAGGTCAAAAGAGGAGTGACGTACATCA
AGGAAAATGTGGATAAACTTATGGAGGGAAATGAGGAGCACATGACTTGTGGGTTCGAAGTGGTGTTTCCGGCGCTTCTACAAAAAGCGAAAAGCTTAGGCATCGAAGATC
TTCCTTACGATTCTCCGGCGGTGCAGGAGGTTTATCATGTCAGGGAACAAAAGTTGAAAAGGATTCCACTGGAGATTATGCACAAAATACCGACATCATTATTATTTAGTTTG
GAAGGGCTCGAAAATTTGGATTGGGACAAACTTTTGAAACTGCAGTCAGCCGACGGTTCCTTCCTCACCTCTCCCTCCTCCACCGCCTTCGCGTTCATGCAAACCAAGGA
TGAAAAATGCTACCAATTCATCAAGAACACGATAGACACTTTCAACGGAGGAGCGCCACACACTTATCCCGTCGACGTGTTTGGAAGGCTCTGGGCGATCGACCGGCTGCA
GCGCCTCGGAATTTCCCGCTTTTTTGAGCCGGAGATTGCTGATTGCTTAAGCCACATCCACAAATTTTGGACGGATAAGGGAGTTTTCAGTGGGAGAGAATCGGAGTTTTG
CGACATTGACGATACATCCATGGGAATGAGGCTTATGAGGATGCATGGATATGATGTTGATCCAAATGTGCTGAGGAATTTCAAGCAGAAAGATGGTAAATTCTCTTGCTACGG
CGGGCAGATGATCGAGTCGCCTTCTCCGATATACAATCTTTACAGAGCTTCTCAGCTCCGATTTCCCGGCGAGGAAATCCTCGAAGATGCGAAGAAATTCGCCTACGATTTC
TTGAAAGAAAAACTAGCCAACAATCAGATTCTGGATAAATGGGTTATTTCTAAGCACTTGCCTGATGAGATCAAGCTCGGGCTAGAGATGCCGTGGCTCGCCACCCTACCCC
GCGTCGAGGCGAAGTACTACATCCAGTACTACGCCGGCTCCGGCGACGTGTGGATCGGGAAGACGCTGTACAGGATGCCGGAGATCAGCAACGACACGTACCACGACCTA
GCCAAGACGGATTTCAAGAGATGCCAAGCGAAGCATCAGTTCGAGTGGCTCTACATGCAAGAATGGTACGAGAGCTGCGGCATCGAGGAATTCGGGATAAGCAGAAAGGA
CCTTCTGCTTTCCTATTTCTTGGCGACCGCGAGCATCTTCGAGCTCGAGAGGACCAACGAGCGAATCGCGTGGGCCAAATCGCAGATCATCGCTAAGATGATCACTTCTTT
CTTCAACAAGGAAACTACGTCGGAGGAGGACAAGCGAGCTCTTTTGAACGAGCTCGGAAACATTAATGGCCTCAACGACACAAACGGCGCAGGGAGAGAAGGTGGGGCC
GGTAGCATTGCGCTAGCGACCCTCACTCAGTTCCTCGAGGGATTCGACAGATACACCAGACACCAGCTGAAAAATGCTTGGAGCGTATGGCTGACGCAGCTGCAACATGGC
GAAGCAGACGACGCAGAGCTCCTAACCAACACGTTGAACATCTGCGCCGGCCACATCGCCTTCAGGGAAGAAATACTGGCGCACAACGAGTACAAAACTCTCTCCAACCTA
ACCAGCAAAATCTGTCGGCAACTTTCTTTCATTCAAAGCGAAAAGGAGATGGGAGTAGAGGGCGAGATCGCAGCGAAATCGAGCATAAAAAACAAGGAACTCGAAGAAGAC
ATGCAAATGTTGGTGAAGTTGGTGCTTGAGAAATATGGGGGCATAGATAGAAATATAAAGAAAGCGTTTTTGGCAGTTGCGAAGACTTATTATTACAGAGCGTATCATGCCGC
CGACACCATAGACACACACATGTTTAAAGTGCTTTTCGAGCCAGTCGCGTGA
After Site-directed mutagenesis：
ATGGCCTCCTTATCCTCTACAATCCTCAGCCGCTCTCCGGCGGCCCGCCGCAGAATTACGCCGGCATCGGCTAAGCTTCACCTGCCGGAATGTTTCGCCATCAGTGCATG
GATGGGCAGCAGCACTAAAAACCTTTCTCTCAGCTACCAACTTAATCACAAGAAAATATCAGTTGCCACAGTAGATGCGCCGCAGGTGCATGACCACGACGGCACTACCGT
TCATCAAGGCCATGATGCGGTGAAGAATATTGAGGATCCCATTGAATACATCAGGACGTTGTTGAGGACGACGGGGGACGGGAGAATAAGCGTGTCGCCGTACGACACGG
CGTGGGTGGCGATGATCAAGGACGTGGAGGGGCGGGACGGGCCCCAGTTCCCCTCCAGCCTCGAGTGGATCGTGCAGAATCAACTCGAGGATGGATCGTGGGGCGATC
AGAAGCTTTTCTGCGTCTACGATCGCCTCGTCAATACCATCGCGTGCGTGGTAGCCTTGAGATCGTGGAATGTTCATGCTCACAAGGTCAAAAGAGGAGTGACGTACATCA
AGGAAAATGTGGATAAACTTATGGAGGGAAATGAGGAGCACATGACTTGTGGGTTCGAAGTGGTGTTTCCGGCGCTTCTACAAAAAGCGAAAAGCTTAGGCATCGAAGATC
TTCCTTACGATTCTCCGGCGGTGCAGGAGGTTTATCATGTCAGGGAACAAAAGTTGAAAAGGATTCCACTGGAGATTATGCACAAAATACCGACATCATTATTATTTAGTTTG
GAAGGGCTCGAAAATTTGGATTGGGACAAACTTTTGAAACTCCAGTCAGCCGACGGTTCCTTCCTCACCTCTCCCTCCTCCACCGCCTTCGCGTTCATGCAAACCAAGGA
TGAAAAATGCTACCAATTCATCAAGAACACGATAGACACTTTCAACGGAGGAGCGCCACACACTTATCCCGTCGACGTGTTTGGAAGGCTCTGGGCGATCGACCGGCTCCA
GCGCCTCGGAATTTCCCGCTTTTTTGAGCCGGAGATTGCTGATTGCTTAAGCCACATCCACAAATTTTGGACGGATAAGGGAGTTTTCAGTGGGAGAGAATCGGAGTTTTG
CGACATTGACGATACATCCATGGGAATGAGGCTTATGAGGATGCATGGATATGATGTTGATCCAAATGTGCTGAGGAATTTCAAGCAGAAAGATGGTAAATTCTCTTGCTACG
GCGGGCAGATGATCGAGTCGCCTTCTCCGATATACAATCTTTACAGAGCTTCTCAGCTCCGATTTCCCGGCGAGGAAATCCTCGAAGATGCGAAGAAATTCGCCTACGATT
TCTTGAAAGAAAAACTAGCCAACAATCAGATTCTGGATAAATGGGTTATTTCTAAGCACTTGCCTGATGAGATCAAGCTCGGGCTAGAGATGCCGTGGCTCGCCACCCTAC
CCCGCGTCGAGGCGAAGTACTACATCCAGTACTACGCCGGCTCCGGCGACGTGTGGATCGGGAAGACGCTGTACAGGATGCCGGAGATCAGCAACGACACGTACCACGA
CCTAGCCAAGACGGATTTCAAGAGATGCCAAGCGAAGCATCAGTTCGAGTGGCTCTACATGCAAGAATGGTACGAGAGCTGCGGCATCGAGGAGTTCGGGATAAGCAGAA
AGGACCTTCTGCTTTCCTATTTCTTGGCGACCGCGAGCATCTTCGAGCTCGAGAGGACCAACGAGCGAATCGCGTGGGCCAAATCGCAGATCATCGCTAAGATGATCACTT
CTTTCTTCAACAAGGAAACTACGTCGGAGGAGGACAAGCGAGCTCTTTTGAACGAGCTCGGAAACATTAATGGCCTCAACGACACAAACGGCGCAGGGAGAGAAGGTGG
GGCCGGTAGCATTGCGCTAGCGACCCTCACTCAGTTCCTCGAGGGATTCGACAGATACACCAGACACCAGCTGAAAAATGCTTGGAGCGTATGGCTGACGCAGCTGCAACA
TGGCGAAGCAGACGACGCAGAGCTCCTAACCAACACGTTGAACATCTGCGCCGGCCACATCGCCTTCAGGGAAGAAATACTGGCGCACAACGAGTACAAAACTCTCTCCA
ACCTAACCAGCAAAATCTGTCGGCAACTTTCTTTCATTCAAAGCGAAAAGGAGATGGGAGTAGAGGGCGAGATCGCAGCGAAATCGAGCATAAAAAACAAGGAACTCGAAG
AAGACATGCAAATGTTGGTGAAGTTGGTGCTTGAGAAATATGGGGGCATAGATAGAAATATAAAGAAAGCGTTTTTGGCAGTTGCGAAGACTTATTATTACAGAGCGTATCAT
GCCGCCGACACCATAGACACACACATGTTTAAAGTGCTTTTCGAGCCAGTCGCGTGA
Three Site-directed mutagenesis:
| None |