Team:SZPT-CHINA/Contribution
Contribution
Characterization of the Anderson Promoter
Characterization of the pR Promoter
References
This year, our team has submitted some new experimental data into existing biobricks. Besides, we
also
advanced the characterization of a few Anderson promoters, which would be a useful tool to other
iGEM
teams in the future.
The parts we used are listed as follows：
The parts we used are listed as follows：
| S/N | Name | Biobricks |
| 1 | J23100 | BBa_J23100 |
| 2 | pR | BBa_R0051 |
● Characterization of the Anderson Promoter
Why did the registry need new information about the Anderson promoter in Gluconacetobacter hansenii ATCC 53582？
In previous studies concerning the Anderson promoter family, the biological chassis used by the iGEM teams was mostly derived from Escherichia coli. Thanks to the well-studied physiology and genetics of this model bacterium, researchers can precisely control gene expression in such a traditional host chassis.
While, in our project a Gluconacetobacter strain was selected as a bacterial chassis since it is a potent producer of cellulose, a group of strong and ultrapure nano-sized biopolymers that are widely used in several industrial domains, including biomedicine, food and textile. In order to optimize bacterial cellulose production in Gluconacetobacter, the open-access constitutive Anderson promoters are employed to modulate the expression of key genes in the cellulose synthesis pathway.
Although the Anderson promoters have been well-studied and widely-used in Escherichia coli, our findings in the literature revealed that the expression pattern in Gluconacetobacter hansenii ATCC 53582 is completely different from that in E. coli. Hereby, we present the characterization of the Anderson promoter family in G. hansenii ATCC 53582, which could be a useful genetic tool in future studies on bacterial cellulose film.
Abstract
Promoter is one of the major tools in synthetic biology, which can be manipulated to tune gene
expression. The strength of a promoter is normally dictated by its precise sequence. Min et al.[1]
investigated transcription performance of a reporter gene mRFP1 that was driven by 11 different
constitutive promoters in G. hansenii ATCC 53582, including the strongest member in the
family
J23119
from an open-access collection known as the Anderson promoter family. The variant promoter
J23119-A27T was
accidentally constructed during the cloning process and it differed from J23119 with only a single
nucleotide at the 27th position. The RBS and terminator in the testing platform were the BioBrick
parts
BBa_B0034 and BBa_B0015
respectively.
(a)
(b)
Figure 1. (a) List of Anderson Promoters that were evaluated in this study.
(b) A schematic depicting the mRFP1 reporter assay used to determine the strength of each candidate promoter.
(b) A schematic depicting the mRFP1 reporter assay used to determine the strength of each candidate promoter.
The expression characteristics of Anderson Promoters in Gluconacetobacter hansenii ATCC 53582
In the literature, the strength of each promoter, as quantified by the fluorescence intensity per
unit
OD600, was normalized to that of the J23119 construct. It is worth noting that these
promoters showed a
bimodal distribution (Figure 2). Among the tested promoters, J23100, J23119-A27T, J23104, J23102 and
J23119 are strong ones. J23119 is the strongest Anderson promoter in E. coli. but not in
G.
hansenii
ATCC 53582. Instead, J23100, J23119-A27T, J23104 and J23102 are consistently stronger than J23119.
Figure 2. Strengths of the various Anderson Promoters in Gluconacetobacter hansenii ATCC
53582
Besides, the strength of a few Anderson promoters in either E. coli or G. hansenii
ATCC
53582 was
compared in the literature. In general, most promoters that are strong in G. hansenii ATCC
53582
also
tend to be strong in E. coli and vice versa. The promoter strength exhibit an apparent
bimodal
distribution pattern in G. hansenii ATCC 53582, whereas they appear to be more evenly
distributed
from 0
to 1 in E. coli.
Figure 3. Anderson promoter strengths in E. coli and Gluconacetobacter hansenii ATCC
53582. The data
for the various promoters were obtained from our mRFP1 reporter assay (Figure 2), while the data for
E. coli were obtained from http://parts.igem.org/Promoters/Catalog/Anderson.
The influence of promoter strength on the sequence characteristics
By comparing the relationship between the strength and sequence characteristics of these 11
promoters
tested in G. hansenii ATCC 53582, we found an interesting correlation. Our data revealed that
the
first
3 bases of the promoter sequence (within the−35 box) have an important role in regulating gene
expression in the Gluconacetobacter genus. The nucleotides "T" (thymine) and "G" (guanine) at
position 1
and 3 respectively seem particularly crucial, as both of them are present in all the strong
promoters,
but absent in all the weak promoters.
For example, J23108 differs from J23119 by only one nucleotide at the first position, but its promoter strength is significantly lower than that of J23119 in all the three strains tested (P < 0.001). Similarly, J23101 differs from J23119-A27T by only a single nucleotide at the third nucleotide position, but its promoter strength is at least 5-fold lower than that of J23119-A27T. In contrast, several nucleotides in the -10 box (underlined in TATAAT) can be mutated without causing a massive effect on the promoter strength.
For example, J23108 differs from J23119 by only one nucleotide at the first position, but its promoter strength is significantly lower than that of J23119 in all the three strains tested (P < 0.001). Similarly, J23101 differs from J23119-A27T by only a single nucleotide at the third nucleotide position, but its promoter strength is at least 5-fold lower than that of J23119-A27T. In contrast, several nucleotides in the -10 box (underlined in TATAAT) can be mutated without causing a massive effect on the promoter strength.
Figure 4. List of Anderson promoter sequence that were evaluated in this study.
● Characterization of the pR Promoter
The cI regulated promoter is based on the pR promoter (BBa_R0051) from bacteriophage lambda. To
compare
the transcriptional strength between the pR promoter and a constitutive promoter J23100 (BBa_J23100),
two plasmids E. coli-DH5α-pR-B0034-sfGFP and E. coli-DH5α-J23100-B0034-sfGFP were
firstly
constructed,
and the reporter gene transcription (Superfolder GFP) controlled by either of two plasmids was
determined by measuring the average fluorescence intensity. To simplify the comparison, we set the
strength of J23100 promoter as 1 and normalized the strength of pR to J23100.
Figure 5. The average fluorescence intensity of the reporter controlled J23100 (E.
coli-DH5α-J23100-B0034-sfGFP) and pR (E. coli-DH5α-pR-B0034-sfGFP).
Excitation is induced at wavelength of 488 nm and the intensity of the emitted fluorescence of
wavelength at 520 nm is measured as a function of transcription strength. Fluorescence is calculated
by
the formula below, LB medium is set as the blank control.
Results
| S/N | Promoter | GFP fluorescence(a.u.) | Proportion |
| 1 | J23100 | 23498.40742 | 1 |
| 2 | pR | 70580.24814 | 3.003618367 |
Figure 6. Fluorescence intensity of reporter gene driven by J23100 (E.
coli-DH5α-J23100-B0034-sfGFP) and
pR (E. coli-DH5α-pR-B0034-sfGFP).
As the figure shows, the expression strength of pR is 3.00 times to that of J23100.
● References
[1] Min Yan Teh, Kean Hean Ooi, Shun Xiang Danny Teo, et al. An Expanded Synthetic Biology Toolkit
for
Gene Expression Control in Acetobacteraceae [J]. ACS Synthetic Biology, 2019, 8(4): 708-723.