Part:BBa_K4583040

PYU92-RBS(B0033)-SRRz

PYU92-RBS(B0033)-SRRz

Usage

We designed a auto-lysis system based on this part. The auto-lysis system will express at the late stationary phase and peaks at about 40h.

Fig. 1 . Genetic circuit of auto-lysis system based on phi X 174 E gene

Late Stationary Phase Promoter

When the bacteria enter the stationary phase, the physiological state of the bacteria changes significantly. During this phase, many genes will respond to make timely adjustments. This 4 parts BBa_K4583000 (PYU3), BBa_K4583001 (PYU7), BBa_K4583003 (PYU16), and BBa_K4583004 (PYU92) are the promoters of E. coil. Their most notable feature is that they will express in the late stationary phase. Moreover, they are self-inducible promoters, which means that no additional inducers are needed to be added for expression. Exogenous inducers are expensive and need to be added artificially, whereas self-induced promoters are cost-effective and relatively stable. This part is also very safe because it comes from E. coli MG1655, a commonly engineered bacterium.

• Late stationary phase promoter
• Self-inducible promoter without additional inducers
• Biosafety

Characterization of Late Stationary Phase Promoter PYU92

Our characterization of this part is divided into two main parts.

• First, this promoter was placed upstream of GFP gene, forming a genetic circuit as shown in Fig. 1. This plasmid was transformed into a bacterium containing another plasmid for characterization. Green and red fluorescence were measured at fixed intervals to compare the expression time and intensity of the two.
• Second, this promoter was placed upstream of the BFP gene, forming a genetic circuit as shown in Fig. 3. This plasmid was then transformed into bacteria containing two other plasmids. Green, red and blue fluorescence were measured at fixed time intervals to compare the difference in expression time and intensity between this part and the other two parts.

For plasmid construction methods and other experimental procedures, see the Design page.

Protocols

Our experimental conditions for characterizing this part were as follows:

• E. coli MG1655
• 30^oC, 48h, under vigorous shaking
• Plasmid Backbone: PACYC
• Equipment: Multi-Detection Microplate Reader (Synergy HT, Biotek, U.S.) and Molecular Devices SpectraMax i3x.

We used GFP (excitation at 485 nm and emission at 528 nm)and BFP (excitation at 400 nm and emission at 450 nm) to characterize this part. As our focus was mainly on the expression time, we processed the obtained fluorescence data by means of the following equation: x'=(x-min)/(max-x). This treatment makes all data fall between 0 and 1, which is easier to use for comparisons between different fluorescence data (since our focus is on expression time).

Characterization using GFP in 2-plasmids bacteria

In this section we used the PACYC plasmid with BBa_K4583004(PYU92) upstream of GFP gene(Fig. 1). We transformed it into L19 and L31 with BBa_K4583009(PesaRwt), BBa_K4583010(PesaRc), BBa_K4583011(PesaRp) plasmids respectively (6 combinations in total) and characterized them using 24-well plates. The characterization results are shown in Fig. 2

Fig. 1 . Genetic Circuit when characterizing PYU92 using GFP

As can be seen from the characterization results, the expression times of this part in most of combinations are significantly different from that of other parts in terms of time and intensity.

We chose two combination(L19-PesaRwt-PYU92 and L31-PesaRwt-PYU92) for the next Characterization.

Fig. 2 . Characterization results of PYU92 in the 2-plasmid bacteria

Characterization using BFP in 3-plasmids bacteria

In this section, we used the PACYC plasmid with PYU7 upstream of the BFP gene (Fig. 3). Based on the results of the last Characterization, we transformed it into L19 and L31 with BBa_K4583009(PesaRwt), BBa_K4583010(PesaRc), BBa_K4583011(PesaRp) and BBa_K4583012(PesaS) plasmid respectively (4 combinations in total) and characterized them using 24-well plates. The characterization results are shown in Fig. 4.

Fig. 3 . Genetic Circuit when characterizing PYU92 using BFP

From the characterization results, we can see that there is a significant delay in the expression of this part from the other promoters. PYU92 is expressed at the stationary phase and peaks at the late stationary phase. We found roughly the same results for both characterizations, but with slightly different onset times. This may be related to the instrumentation used. For this characterization, we used a Molecular Devices SpectraMax i3x, which has a much higher precision. In addition, the difference between the 2-plasmids system and the 3-plasmids system may also account for the difference.

Fig. 4 . Characterization results of PYU92 in the 3-plasmids bacteria

Auto-lysis system

We ligated the SRRz gene to the plasmid backbone we constructed by Gibson's method to obtain PACYC-PYU3-SRRz,PACYC-PYU7-SRRz,PACYC-PYU16-SRRz,PACYC-PYU92-SRRz plasmid(Fig. 3).

Fig. 6 . Plasmid construction

Fig. 7 . Lysis effect of SRRz gene

In order to enhance its lysis effect in L19 and L31 strains, we decided to do so by increasing the RBS of the promoters of PYU3,PYU7,PYU16. We added RBS named B0031,B0032,B0033,B0034 with four different intensity gradients with RBS intensity of 0.01,0.07,0.3,1. A second experiment was performed.

Due to time and effort constraints, we did not succeed in constructing all plasmids. The strain that we successfully construct are as follows:

\

No.	Strains	Backbone	Promoter	RBS	Lysis gene
1	L19	PACYC	PYU3	B0031	SRRz
2	L19	PACYC	PYU3	B0032	SRRz
3	L19	PACYC	PYU16	B0031	SRRz
4	L19	PACYC	PYU16	B0034	SRRz
5	L31	PACYC	PYU92	B0031	SRRz
6	L31	PACYC	PYU92	B0032	SRRz
7	L31	PACYC	PYU92	B0034	SRRz

We then proceeded to characterise these strains by placing them in Multi-Detection Microplate Reader (Synergy HT, Biotek, U.S.) to determine the OD600. We found that only two combinations were able to reduce their OD600 values.

Analysis of our results

Although we have only been able to prove the effectiveness of two systems (PesaS-B0034-PYU16-B0034-SRRz and PesaS-B0034-PYU92-B0034-SRRz ), we have learned a lot from them. During culturing a large amount of cellular debris is produced using the lysis system. This can interfere with the detection of OD600. This is probably why most systems "don't work": the debris blocks the light path and the OD600 does not reflect the number of viable bacteria.We propose several possible solutions.

• Enumeration by live cell counting other than OD600.
• Counting by spread plate method.
• Allow the solution to stand for a period of time (15-30 min) and then collect the supernatant to measure OD600. The data obtained will be different from the true value but may reflect the lysis situation.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

Reference

[1] Gao, Y., et al., Inducible cell lysis systems in microbial production of bio-based chemicals. Appl Microbiol Biotechnol, 2013.

[2] Talukder, A.A., et al., RpoS-dependent regulation of genes expressed at late stationary phase in Escherichia coli. FEBS Lett, 1996. 386(2-3): p. 177-80.

[3] Gu, F., et al., Quorum Sensing-Based Dual-Function Switch and Its Application in Solving Two Key Metabolic Engineering Problems. ACS Synth Biol, 2020. 9(2): p. 209-217.