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−	We then proceeded to characterise these strains by placing them in xx to determine the OD600. We found that only two combinations were able to reduce their OD600 values.	+	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===		===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.		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.

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Revision as of 13:35, 9 October 2023

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Name: SRRz lysis cassette

Base Pairs: 1650 bp 

Origin: Lambda phage

Properties: Bacterial cell lysis

Implementation and function

Being one of the most widely studied bacterial mechanisms, bacterial cell lysis can be evaluated through the expression of native lyric proteins within the cell. Lysis mechanism could be attained and exploited for designed usages, such as promoting cellular membrane disruption, or acting as an intermediate action to release certain proteins to extracellular solutions. Lysis of bacterial hosts or bacterial walls is deliberately scheduled and regulated, accumulating lysozyme activities. In this basic part, we emphasize the use of the SRRz/Rz1 lambdoid lysis cassette, which consists of a lysin and a holin gene.

At first, the T7 constitutive promoter transcribes and translates β-galactosidase and stores the enzyme in the cell. When testing is about to process, the samples are added to the straining container. The promotor causes the ribosomal binding site to initiate the lysis of the gene, causing the bacterial wall to dissolve and releasing the β-Galactosidase.

SRRz lysis cassette, as well as other lysis gene sequences, are recognized for their importance in cell disruption techniques for attaining specific intracellular proteins. One example of a mechanical technique is cell ultrasonication, which often results in protein denaturation, due to the heat produced during the process. As an alternative to mechanical techniques, chemical techniques including membrane decadence resulting from lysozyme activities, can be considered in use. Lysis systems could be engineered and targeted for the recovery or replacement of intracellularly expressed proteins. 

Procedures to prove our lysis module using copper-sensitive promoter:

Cultivation

Firstly, add liquid LB to a tube or flask and add the appropriate strain to the correct concentration. Then sing a sterile pipette tip to select a single colony from your LB agar plate and loosely cover the culture with a cap. Incubate the E. Coli at 37 degrees Celsius and 200 rpm so that the beta galactocidase to function in its optimal condition. After incubation, use OD600 to measure the density of the culture. When OD600 values equals to 0.3, add different concentrations of arabinose. Measure OD600 values at 0.5h, 1h, 1.5h, 2h, 3h and 4h intervals.

Prove

The results suggested that the lysis circuit works regularly when the concentration of copper ions is above 10^-6 molL. The rapid decline of OD600 at 10^-5 molL indicates lysis of bacterial wall, which proves that our lysis module could function normally and continue to work in a relevant context.

Lysis Module

We used the copper-sensitive CusR/CusS promoter, a two-component signal transduction system that is responsive to copper, coded by Escherichia coli. This nucleotide sequence is believed to be able to bind with phosphorylated CusR transcription factor in E.coli. CusR protein is phosphorylated by CusS transmembrane protein in a case of high extracellular concentration of copper ions. After phosphorylation CusR interacts with described DNA sequence and activates the transcription of CusA, CusB, CusC and CusF genes coding the proteins of the copper metabolic system. This system is initiated by the expression of pcoE, induced by copper ions. pcoE is a gene from the copper-resisting operon pco in. E.coli. Similar to CusR and CusS, PcoR and PcoS from the pco operon are two-component systems, which are also involved in regulating metal-responsive genes.

The SRRz gene codes maybe three proteins: S,R,Rz. The product of S gene would cause lesions on the cytoplasmic membrane through which the product coded by the R gene escapes to the periplasm and causes murein-degrading, while the Rz gene’s product may be an endopeptidase that can cleave the oligopeptide crosslinks in the peptidoglycan and/or between peptidoglycan and the outer membrane.

Procedures to prove our lysis module using lac promoter:

1. Cultivate E. Coli in LB mediums at 37 degrees Celsius and 200 rpm.

2. When OD600 values equals to 0.4, add different concentrations of copper, three times for each group.

3. Measure OD600 values at 0.5h, 1h, 1.5h, 2h, 3h and 4h intervals.

the graph suggested that the lysis circuit works regularly when the concentration of copper is above 10^-5 mol/L.

Our system is initiated by two components: pBad/araC promoter (BBa_I0500) and copper-sensitive promoter (BBa_I760005). Being two efficient and stable promoters, they induce the expression of lysis genes inserted in the bacterial plasmids productively, guaranteeing the working efficiency of our lysis module.

Procedures to prove our lysis module using pBad/araC promoter:

1. Cultivate E. Coli in LB mediums at 37 degrees Celsius and 200 rpm.

2. When OD600 values equals to 0.3, add different concentrations of arabinose, three times for each group.

3. Measure OD600 values at 0.5h, 1h, 1.5h, 2h, 3h and 4h intervals.

The results suggested that the lysis circuit works regularly when the concentration of arabinose is above 10^-6 mol/L. The rapid decline of OD600 at 10^-5 mol/L indicates lysis of bacterial wall, which proves that our lysis module could function normally and continue to work in a relevant context.

Procedures to prove our lysis module using copper-sensitive promoter:

1. Cultivate E. Coli in LB mediums at 37 degrees Celsius and 200 rpm.

2. When OD600 values equals to 0.3, add different concentrations of copper, three times for each group.

3. Measure OD600 values at 0.5h, 1h, 1.5h, 2h, 3h and 4h intervals.

Reference

Leuzzi A, Grossi M, Di Martino ML, Pasqua M, Micheli G, Colonna B, Prosseda G. Role of the SRRz/Rz lambdoid lysis cassette in the pathoadaptive evolution of Shigella. Int J Med Microbiol. 2017 Jun;307(4-5):268-275. doi: 10.1016/j.ijmm.2017.03.002. Epub 2017 Mar 28. PMID: 28389211.1

Pasotti L, Zucca S, Lupotto M, Cusella De Angelis MG, Magni P. Characterization of a synthetic bacterial self-destruction device for programmed cell death and for recombinant proteins release. J Biol Eng. 2011 Jun 7;5:8. doi: 10.1186/1754-1611-5-8. PMID: 21645422; PMCID: PMC3127821.

Munson GP, Lam DL, Outten FW, O'Halloran TV. Identification of a copper-responsive two-component system on the chromosome of Escherichia coli K-12. J Bacteriol. 2000 Oct;182(20):5864-71. doi: 10.1128/JB.182.20.5864-5871.2000. PMID: 11004187; PMCID: PMC94710.

Young R. Phage lysis: three steps, three choices, one outcome. J Microbiol. 2014 Mar;52(3):243-58. doi: 10.1007/s12275-014-4087-z. Epub 2014 Mar 1. PMID: 24585055; PMCID: PMC4012431.

srrz cell lysis gene

The SRRz gene codes maybe three protein S,R,Rz.The product of S gene would cause lesions on the cytoplasmic membrane through which the product coded by the R gene escapes to the periplasm and causes murein-degrading, while the Rz gene’s product may be an endopeptidase that can cleave the oligopeptide crosslinks in the peptidoglycan and/or between peptidoglycan and the outer membrane.

Sequence and Features

Characterization and improvement contribution made by iGEM23_SDU-CHINA

Group: iGEM 2023 SDU-CHINA

Author: Suiru Lu and Chao Tang

Summary: 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.

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.

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 PYU16 (an example)

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.

1. 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).

2. Characterization using GFP in 2-plasmids bacteria

In this section we used the PACYC plasmid with BBa_K4583003(PYU16) 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

Only one combination showed significant differences in expression time and expression intensity (L31-PesaRp-PYU16).

3. 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_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.

From the characterization results, we can see that there is a significant delay in the expression of this part from the other promoters. PYU16 is expressed at the stationary phase and peaks at the late stationary phase (42h). 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.

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).

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.