From 2007.igem.org

09/24/07

• Ligation for [http://partsregistry.org/Part:BBa_I763031 I763031].
• Transformation of [http://partsregistry.org/Part:BBa_I763031 I763031].
• We inoculate [http://partsregistry.org/Part:BBa_I763036 I763036], [http://partsregistry.org/Part:BBa_I763007 I763007], [http://partsregistry.org/Part:pSB4A3 pSB4A3] in 5ml of LB.
• Transformation of [http://partsregistry.org/Part:BBa_I763028 I763028].

09/25/07

• Miniprep of:

- [http://partsregistry.org/Part:BBa_I763036 I763036];

- [http://partsregistry.org/Part:BBa_I763007 I763007];

- [http://partsregistry.org/Part:pSB4A3 pSB4A3];

- [http://partsregistry.org/Part:BBa_I763033 I763033].

• We inoculate (5ml LB, 5ul amp 1000X) straked transformations of:

- [http://partsregistry.org/Part:BBa_I763033 I763033];

- [http://partsregistry.org/Part:BBa_I763028 I763028] (vitality test).

• Transformations of:

-[http://partsregistry.org/Part:BBa_I763031 I763031].

09/26/07

Fluorescence test according to Fluorescence Test Protocol of:

1. [http://partsregistry.org/Part:BBa_I763004 I763004]:

-bacteria use to have the PMT output maximum value;

-normalization of OD value at 0.5;

-output value without IPTG: [0.22, 0.27]V;

-output value with IPTG:

-after 10min: [0.18,0.28]V;

-after 20min: [0.41,0.46]V;

-after 30min: [0.42,0.70]V;

-after 60min: [0.48,0.80]V;

-thus we decide to grow the OD value to have higher output value.

2. [http://partsregistry.org/Part:BBa_I763028 I763028]

- we observe bacteria vitality, but there isn’t fluorescence after O/N growth with and without progressive induction with IPTG (1mM, 2mM, 3mM, 10mM every 60min).

- thus we decide to use lactose.

09/27/07

Fluorescence test according to Fluorescence Test Protocol of:

1. [http://partsregistry.org/Part:BBa_I763004 I763004]:

-output value like to value with OD=0.5.

2. [http://partsregistry.org/Part:BBa_I763028 I763028]:

-growth in LB medium;

-pellet in M9 with lactose;

-we observe them every 30min:

-bacteria vitality confirmed;

-bacteria don’t beam fluorescent.

3. [http://partsregistry.org/Part:BBa_I763031 I763031]:

-Output value without IPTG:0.05V;

-Output value with IPTG: about 0V.

4. [http://partsregistry.org/Part:BBa_I763019 I763019]:

-Output value without IPTG:0.05V;

-Output value with IPTG: about 0V.

Conclusions:

-using of low copy plasmid;

-higher OD;

-[http://partsregistry.org/Part:BBa_I763032 I763032] and [http://partsregistry.org/Part:BBa_I763034 I763034] fluorescent test.

• We inoculate [http://partsregistry.org/Part:BBa_I763033 I763033] and [http://partsregistry.org/Part:BBa_I763028 I763028].

09/28/07

• Digestion of : [http://partsregistry.org/Part:BBa_I763033 I763033] with Eco/Spe.
• Ligation with [http://partsregistry.org/Part:BBa_J04431 J04431] (Eco/Xba).
• We transform and strake on plates.

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