Contribution
Overview
The poor efficiency and high cost of levan's
detection and production are meaningful and urgent to be solved, due to the fact that levan is
discovered with such good and wide range of use in meditation and others that it shows a massive future
market prospect, which have mentioned above. So as to solve both of these issues, our team designed six
plasmids. In the constructing of the first and second plasmids, we added levan gene and its controlling
gene into the empty plasmid we are using: pUC19. During the construction of plasmid three and four, we
added RFP gene and its controlling gene into our plasmid via homologous recombination, which after these
steps, the levan fluoresced red that people can easily identify it by human eyes and also precisely by
machines. In an effort to increase productivity of levan even further, we keep on constructing the fifth
and sixth plasmids. We tested two different promoters: plac and puv5 which liked the motor of levan and
the results showed that the sixth plasmid exhibited an advance in productivity.
Table 1. Part Collection
|
Parts No.
|
Parts Name
|
Contribution type
|
Part type
|
|
BBa_K4873001
|
zliE-zliS
|
basic part
|
Regulatory
|
|
BBa_K4873002
|
pUC19
|
basic part
|
Plasmid_Backbone
|
|
BBa_K4873003
|
pUCzliES
|
composite part
|
Plasmid
|
|
BBa_K4873004
|
sacB
|
basic part
|
Reporter
|
|
BBa_K4873052
|
pUCzli-SacB
|
composite part
|
Plasmid
|
|
BBa_K4873006
|
RFP
|
basic part
|
Reporter
|
|
BBa_K4873054
|
pUCzli-SacB-RFPT
|
composite part
|
Plasmid
|
|
BBa_K4873007
|
pzliE
|
basic part
|
Regulatory
|
|
BBa_K4873055
|
pUCzli-SacB-PRFPT
|
composite part
|
Plasmid
|
|
BBa_K4873008
|
Plac
|
basic part
|
Regulatory
|
|
BBa_K4873056
|
pUCzli-PlacsacBRFP
|
composite part
|
Plasmid
|
|
BBa_K4873009
|
PlacUV5
|
basic part
|
Regulatory
|
|
BBa_K4873057
|
pUCzli-PUVsacBRFP
|
composite part
|
Plasmid
|
Create New Parts
1.
A new contribution part
pUCzli-SacB-RFPT （
BBa_K4873054
）
RFP was constructed into
pUC-zli-SacB
, however, this plasmid cannot express RFP as it lacks the corresponding
promoter sequence.
Fig.1 Sequencing construction of pUC-Zli-SacB-RFP
2.
A new contribution part
pUCzli-SacB-PRFPT （BBa_K4873055
）
This part added a promoter to RFP, allowing RFP to express.
Fig.2
Sequencing results indicate the successful
construction of pUC-Zli-SacB-PRFPT
3.
A new contribution part
pUCzli-PlacsacBRFP (
BBa_K4873056
)
C
ompared to pUCzli-SacB-PRFPT （BBa_K4873055
）
, replaced the promoter sequence naturally carried by
SacB with plac (a different promoter sequence), in the hope of achieving improved expression of
SacB.
Fig.3 Sequencing results indicate the successful construction of
pUC-zli-plac-SacB-PRFP
4.
A new contribution part
pUCzli-PUVsacBRFP (
BBa_K4873057
)
Compared to
pUCzli-PlacsacBRFP (
BBa_K4873056
)
, replaced the plac promoter with the puv promoter, altering the promoter,
to compare the difference in expression of SacB.
Fig. 4 The sequencing results confirm the successful construction of
pUC-zli-puv-SacB-PRFP
Functional Test
In order to investigate the expression levels of SacB in different promoter
systems (
pUCzli-SacB-PRFPT
,
pUCzli-PlacsacBRFP
,
pUCzli-PUVsacBRFP
), we first compared the fluorescence intensity of RFP. We observed that in
the
pUCzli-PlacsacBRFP system, SacB exhibited the highest
red fluorescence intensity. Subsequently, we measured the fluorescence intensity values of RFP,
which also confirmed that pUCzli-PlacsacBRFP had higher values, indicating that SacB has a higher
expression level .
Fig.5 In these expression systems, when comparing the brightness of RFP,
pUCzli-PlacsacBRFP exhibits stronger RFP expression
References
1. Feng, J., Gu, Y., Quan, Y. et al. (2015). Recruiting a new strategy
to improve levan production in Bacillus amyloliquefaciens. Sci Rep 5, 13814
2. Cancer - World Health Organization(WHO). (2020) the key facts about
cancer. (who.int )