Team:Kyoto/Project/Goal B

Goal B: Characterization of λ Lysis cassette

Introduction

λ Lysis cassette is a gene that causes cell lysis. In previous iGEM, some teams evaluated the lytic activity of λ Lysis cassette qualitatively. However, when we considered λ Lysis cassette not only as a device of cell lysis but as a device of cell death, we must study about λ Lysis cassette more quantitatively. For example, we must study about when cells die, how many cells die, and the relationship between them and the activity of their promoter.

Construction

To characterize the lytic activity of Λ Lysis cassette quantitatively, we regulate the gene expression of λ Lysis cassette by a lactose promoter, R0011, which we characterized quantitatively by RPU in GoalA. To repress the basal expression of λ Lysis　cassette without IPTG as possible, we used pSB4K5, a low copy vector, as a plasmid backbone and used KRX as a host cell. Needless to say, we characterized R0011 in the same experimental condition. For more detailed explanation of the characterization of R0011, go Goal A

Result

Experiment 1 Mesurement of lytic activity of λ　Lysis cassette

To characterize the lytic activity of λ Lysis cassette quantitatively, we made another mesurement standared of cell lysis. we considered that we could characterize the lytic activity of λ lysis cassette by mesuring A550 of the cultures after induction of IPTG.However, we did't know the nature of it, so we couldn't characterize λ Lysis cassette quantitatively if we cheese the best timing of mesurement. As we did some experiences, we earned some facts by try and error. ・Cultures induced the expression of λ　Lysis cassette becomes a kind of equilibrium state of cell lysis. ・R0011 is very easy to have mutation at 37 if it regulates expression of toxic gene because of natural selection. ・The possibility of mutation become low at 30, although わずかな変異の可能性はあるが

Therefore, we made a mesurement standard that is suitacle to our experiment.

Pick out three colonies that is transformed with and cultivated in suppemented M9 media with various concentration of IPTG.After incubation of 16h,18h,20h, mesure A550 of each culture.

We did the experiment as this protocol, we got the following data.

Table 1 A550 of the cultures incubated 16h, 18h, 20h, in various concentration of IPTG.

As table1, the cultures surely get to the equilibrium state of cell lysis depending on the cocentration of IPTG, and we considered that the number of A550 have a quantitative of the lytic activity. We adapted to the data of 18h.

Fig.1 A550 of 18h cultures with various concentration of IPTG. When the concentration of IPTG is from 0.01mM to 0.02mM, A550 decreases mildy. When the concentration of IPTG is from 0.02mM to 0.05mM, A550 decreases dramatically. When the concentration of IPTG is over 0.05mM, A550 is under 0.3 and doesn't change largely.

To analize the relationship between the lytic activity of λ Lysis cassette and RPU, we used the result of characterization of R0011.

Fig.2 Fig.2 is the figures that the horizontal axis　of Fig 1 is converted to RPU using the result of characterization of R0011 りんくよろ. RPU is the absolute activity of promoter, so Fig2 shows the relationship between the lytic activity of λ Lysis cassette and the induction of λ Lysis cassette.

Fig２の結果から、RPUが　　　のときはK は確かに働かず、溶菌しない。ＲＰＵが　　になると、若干positivecontolと比べてA550が減少っするため、細胞死が起きている。RPUが　　以上になると溶菌平衡が下がっていき、ＲＰＵが をこえたところで大きな変化はなくなり溶菌はMAXになる。以上の結果から、λ Lysis cassetteは以下のようにワークするとかんがえられる

Experiment 2 Measurement of lytic activity with time

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Discussion

Equilibrium state of cell lysis

われわれの実験結果から、λLysis cassetteを使用した細胞死というのはinductionに依存した一種の平衡状態になるとかんがえられる。 しかし、ＲＰＵとの対応データからでは、lytic activityは0.2RPU以上ではあまり変化しない。この減少を説明するために、　以下の三つのモデルを考えてみた 1　カルチャーの中である細胞死のサイクルが起きている 2　エンドライシンが細胞壁を分解するのに限界がある 3 λ　Lysis cassette の発現量の限界 詳しくはモデルで

Possibility of a system controlling cell populatiuon

λ　Lysis cassetteのinductionによって細胞溶菌平衡状態に達し、一定の細胞数となることを利用して、cell populationを制御できるはず　それって今後のシンセティックバイオロジーに役立つのでは？　これはfutureかも

Checkpoint of whether cell lysis occurs

λ　Lysis cassette を何かのプロモーターによって制御しシグナル依存的な細胞死(細胞溶菌)を達成したい場合、プロモーターのbasal activity が RPU以下でなくては安定した系をつくることはできないとかんがえられる

iGEMのプロモーターでＲＰＵデータあるもののbasalと比較し、これは使える　使えないを判別？

Initiation of cell lysis

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References

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