Difference between revisions of "Part:BBa K3790014"

Latest revision as of 16:33, 21 October 2021

The simplest form of dps promoter, a strong σS promoter

Introduction

RNA polymerase in E. coli. relies on combined with σ factors to recognize promoter sequences. Although σ70 factor is responsible for the transcription of most of the genes in the genome. E. coli has other six kinds of alternative σ factors that are triggered in stressful conditions. By switching σ factors, bacteria can dramatically change the whole transcription pattern in order to express specific proteins to help themself survive in a bad day.

Among the six alternative σ factors, σS factor recognizes DNA sequences that are very similar to the typical promoter sequences recognized by σ70. Therefore, it is thought that σS promoters rely on upstream elements^[1], which are protein-binding DNA sequences distributed upstream, to show their dependence on σS factors.

We noticed that there’s no standard nor well-characterized σS promoter in the registry. Therefore, we tested the core regions of several σS promoters and examined where they can be recognized by σS factors independently. Among them, the core region of the promoter controlling the expression of dps in E. coli became our favorite, shows highest strength as a σS promoter.

Usage and Biology

The sequence of this part shows high transcription promoting activity in stressful conditions, such as carbon starvation. Typically, the expression of protein regulated by this part will be dramatically enhanced when bacterial growth reaches a plateau. At that time, σS can help efficiently expressing proteins when the density of bacteria in culture is super high.

WT T7 phage naturally has a gene product (gp) 5.7 (Z0141, K3790050), that acts as an efficient inhibitor of σS-dependent transcription. We image we could develop a circuit simultaneously controls the expression of all the proteins whose transcription is regulated by the expression of gp5.7.

Worth to Notice

This part is the simplified form of dps promoter. It’s short and functions as a σS promoter independently. It can be freely fused with other regulatory parts, such as operators, to create parts with novel properties.

Characterization

Growth Curve and GFP Concentration

Plasmids (Figure 1,2) were constructed and respectively transformed into DH5α E. coli. Three independent colonies of each strain were picked out and cultivated at 37℃ overnight in LB culture medium with ampicillin. Then, the bacteria solution was diluted to 1% with 100 ml LB culture medium and shaked for 11.5 hours in 37℃. Samples of bacteria solution were collected every 0.5 hour, and then stored in 4℃ for measurement later using a fluorescent plate reader. After 11.5 hours, all the samples were transfered from eppendorf tubes into wells on 96-well plates. OD600 and fluorescence intensity (excitation: 488 nm, emission: 530 nm) of each sample was measured twice with a plate reader.

Figure 1. GFP controlled by dps promoter (pdps). The RBS is part B0034. The GFP is part E0040. The terminator is part BBa_0015.

Figure 2. GFP in J364001 is driven by J23106, a constitive promoter.

All the measurements of fluorescence intensity were standarized by the following standard curve (Figure 3).

Figure 3. Fluorescein solutions with known concentrations were used for GFP standarization.

As shown in Figure 4, the OD600 (1 OD equals to 10^8 950-nm diameter silica nanoparticles from NanoCym) growth curves of both bacteria share similar trend. Both bacterial strains are at exponential stage before 5 hours in culturre, and then reach a plateau. The overall OD600 curve from the strain with pdps driven GFP expression is slightly higher then the one with σ70 promoter driven GFP (the black line, the control group). This is consist with that most of the genes relevant to rapid growth in E. coli are regulated by σ70 factors, and pdps initiates the transcription occupying less σ70 factors then σ70 promoter dose.

Figure 4. Growth curve of bacteria transformed with plasmids shown in Figure 1 and Figure 2.

Interestingly, the GFP/OD600 of the control group rises during exponential growth, but stop increasing after reaching plateau (Figure 5). The experimental group features much bigger increasing rate of GFP/OD600 during platform stage then exponential stage. This can be explained by the fact that σ70 is the major σ factors during exponential stage in E. coli, while σS takes the dominance during platform stage for carbon starvation.

Figure 5. Calibrated GFP per OD during growth, of strains transformed with plasmids shown in Figure 1 and Figure 2.

Compare six σS promoters

Six plasmids with different σS promoters regulating the expression of GFP (similar to Figure 1) were compared, after 11.5 hours cultivation (Figure 6).

Figure 6. Amount of GFP per OD of six σS promoters after 11.5h of cultivation. The six σS promoters are respectively K3790009, K3790010, K3790011, K3790012, K3790013, k3790014.

It can be seen in Figure 6 that, among these six σS promoters, pdps shows the highest strength.

By the way, the suffix _mut involved a single nucleotide substitution (C to be specific) at -13 location of the original promoter. This substitution could enhance the strength of σS promoters^[2]. There is a C at -13 location of original pdps.

T7 gene product 5.7 inhibition test

Gene product 5.7 is a gene expressed during the T7 infection mid-stage. It's a protein that can bind to σS factors and RNA polymerase of E. coli, to inhibit the transcription initiated by σS-RNAP complex^[3].

Figure 7. Plasmid for inducible T7 gp5.7 expression.

We constructed the plasmid shown in Figure 7, T7 gp5.7 regulated by araC/pBAD. Co-transform it with the plasmid shown in Figure 1 (GFP driven by pdps) into DH5α E. coli. We measured the growth curve and GFP concentration of this strain after different concentrations of arabinose induction.

Figure 8. GFP per OD during bacterial growth, after different concentrations of arabinose induction.Time zero point was when arabinose was added.

Arabinose induction could activate the expression of T7 gene product 5.7 (Figure 7), which could inhibit σS promoter, including pdps. As is shown in Figure 8, the expression of GFP regulated by pdps is decreased, as expected, after arabinose induction. We are still investigating why the decrease did not correlate with arabinose concentration. We have built a mathmatic model and use simulation to help us understand this process https://2021.igem.org/Team:Fudan/Model

Sequence and Features

References

1. ↑ What makes an Escherichia coli promoter sigma(S) dependent? Role of the -13/-14 nucleotide promoter positions and region 2.5 of sigma(S). Becker G,  Hengge-Aronis R. Mol Microbiol, 2001 Mar;39(5):1153-65. PMID:11251833
2. ↑ In vitro transcription profiling of the σS subunit of bacterial RNA polymerase: re-definition of the σS regulon and identification of σS-specific promoter sequence elements. Maciag A,  Peano C,  Pietrelli A,  Egli T,  Bellis GD,  Landini P. Nucleic Acids Res, 2011 Jul;39(13):5338-55. PMID:21398637
3. ↑ T7 phage factor required for managing RpoS in Escherichia coli. Tabib-Salazar A,  Liu B,  Barker D,  Burchell L,  Qimron U,  Matthews SJ,  Wigneshweraraj S. Proc Natl Acad Sci U S A, 2018 Jun 5;115(23):E5353-E5362. PMID:29789383