Team:Shanghai HS/Parts
Basicparts
Promoter: BBa_J23100
Description: Parts J23100 is included in a family of constitutive promoter parts isolated from a small combinatorial library.This part presents in plasmid J61002 and the RFP is at the downstream of the part.These promoter parts can be used to tune the expression level of constitutively expressed parts. The reason we employed this promoter is that it is well developed and easy to reconstruct: the NheI and AvrII restriction sites within these promoter parts make them a foundation for further modification.
Bba_B0034
It is a kind of RBS that we used. a sequence of nucleotides upstream of the start codon of an mRNA transcript that is responsible for the recruitment of a ribosome during the initiation of protein translation.
MlrA2s
Description: This part has the similar effect as MlrA2. It can help us to make the microcyctin antitoxic. This part is derived from a specie called Novosphingobium sp. THN1 based on a paper in China. And we also modified the codon inside the gene in order to make it fit the vector (BL21DE3, DH5α) we employed and there for we the protein will be better expressed.MlrA3 is from Novosphingobium sp. THN1(NCBI: AEC46646.1).
6x his tag
Description: We add 6xHis tag into the coding region by PCR. Facilitating purification, 6x His tag severs as a marked purification tag for Ni-NTA column. Moreover, 6xhistag also helps expressed enzymes immobilize as well as bind to our designed prototype device.
BBa_B0015
Description: The mfold results are annotated with the location of the subparts BBa_B0010 and BBa_B0012 and the BioBrick assembly scar. Double terminator consisting of BBa_B0010 and BBa_B0012.This is the most commonly used terminator. It seems to be reliable. However, that Part:BBa_B0014 is a better part for forward and reverse termination.
Sumo
Description: It is a cleavable ubiquitin-like protein tag. It increases the solubility of our product. Since it is easy to be expressed, we use it to make our protein have a better expression. We tried to express our protein without Sumo tag but we failed to do so.
T7 promoter
Description: It is a strong promoter. Its inner structure is conservative, meaning that it can combine with most of the protein and therefore it would make the translation better off. Such feature can make it a universal promoterT7terminator
Description: It is a strong terminator. Similar to T7 promoter, its unique structure make it easy to combine with most of the protein and thus it is safe to use.
TEV site
Description: Initially we design to employ it in order to cut the SUMO tag out: after the expression of protein, SUMO tag became useless and may influence the function of our protein. Yet, we found out that once we cut the SUMO tag off, our protein will deposit and lose all its function.Reference
[1] Active and silent members in the mlr gene cluster of a microcystin-degrading bacterium isolated from Lake Taihu, ChinaComposite parts
pSB1C3-MlrA2
T7 promoter
Description: It is a strong promoter. Its inner structure is conservative, meaning that it can combine with most of the protein and therefore it would make the translation better off. Such feature can make it a universal promoterT7terminator
Description: It is a strong terminator. Similar to T7 promoter, its unique structure make it easy to combine with most of the protein and thus it is safe to use.TEV site
Description: Initially we design to employ it in order to cut the SUMO tag out: after the expression of protein, SUMO tag became useless and may influence the function of our protein. Yet, we found out that once we cut the SUMO tag off, our protein will deposit and lose all its function.Reference
[1] Active and silent members in the mlr gene cluster of a microcystin-degrading bacterium isolated from Lake Taihu, ChinaComposite parts
pSB1C3-MlrA2pSB1C3 is the first composite part we employed during our experiment. It is composed of BBa_J23100, BBa_B0034, MlrA2, MCS and 6xHistag. BBaa_J23100 is a well-known promoter. We use it since its features are well understood and easy to be modified. BBa_B0034 is a kind of RBS. It is a sequence of nucleotides upstream of the start codon of an mRNA transcript that is responsible for the recruitment of a ribosome during the initiation of protein translation. MlrA2 is the core part of our experiment. It is responsible for the degradation of microcyctin. MCS is the part that carried by our device and 6xHistag can help us to combined our protein with the Ni column pSB1C3-MlA3
Similar to pSB1C3-Mlra2, it is the composite part we deigned and used at the beginning of our experiment. We replace the MlrA2 inside pSB1C3-MlrA2 by MlrA3 to create this part. It has the same structure with MlrA3 and can help us get the target protein that can react with Microcystin.
pET28a-MlrA2
This is another composite part we used during our experiment. It is composed of T7 promoter, lac operator, RBS, MlrA2, MCS, 6xHistag and T7 terminator. T7 promoter is a strong promoter. Its structure is so conservative that it can successfully combine with most of the protein. MlrA2 is the core part that we designed and it is aimed to degrade Mycrocystin. 6xHistag can help us to combined the target protein with the Ni column during the purification of the protein. And the same as T7 promoter, the unique structure of T7 terminator allow it to safely combine with most of the protein and thus is an efficient, universal terminator. pET28a-MlrA3
Instead of putting MlrA2 into the device, like pET28a-MlrA2, we put MlrA3 into the device, creating a new composite part. The same as pET28a-MlrA2, we used T7 promoter, lac operator, RBS, MCS, 6xHistag and T7 terminator. The new added MlrA3 would allow us to create a new protein that can efficiently degrade Mycrocystin.
pTolo-EX5-MlrA2
pTolo-EX5 is the composite part we employed to create our final products. It is composed of T7 promoter, RBS, 6xHis tag, SUMO tag, TEV site, MlrA2 and T& terminator. The main different between this part and the previous one is that we added SUMO tag and TEV site into the device. At first, we added SUMO tag into the device in order to increase the solubility of our protein. We expect the protein to be better purified through the use of SUMO tag. We then added TEV site, which allow us to cut off the SUMO tag after purification. We worried that the SUMO tag will influence the effect of our protein. Yet, we find that it is impossible to get rid of the SUMO tag: one it is cut off, the protein well deposit immediately. Fortunately, carrying SUMO tag didn’t impact the result of our experiment. pTolo-EX5-MlrA3
The new added MlrA3 gene allows us to create a new protein to handle the Mycrocystin. pTolo-EX5-MlrA3 is composed of T7 promoter, RBS, 6xHis tag, SUMO tag, TEV site, MlrA3 and T& terminator. The strong promoter and terminator, along with the SUMO tag, make it an incredible weapon against the toxic substance, Mycrocystin, that cyanobacterial contains.
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