Dear Switch/Count team members,

In this email will be a brief statement of our current situation. It's meant to be a reference that you may want to look at while you are in the lab, as well as a stimulus for discussion while you are absent.

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* where we are are head for
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Our present road map can be summarized as below:

Step 1a. PCR promoter cI434OR21 & cI434OR321 (noted as 434OR), assemble it into the switch plasmid (pSB3K3 from I7100, Kan+).
Product: 434OR on pSB3K3

Step 1b. PCR gene cI, assemble it into the switch plasmid obtained above.
Product: cI_434OR on pSB3K3

Step 2a. PCR gene EGFP, assemble it into the the switch plasmid.
Product: EGFP on pSB3K3

Step 2b. PCR gene cI434, assemble it into the switch plasmid obtained above.
Product: cI434_EGFP on pSB3K3

(Step 1 and 2 will go in parallel.)

Step 3. Cut cI_434OR from the product of Step 1b, assemble it into the product of Step 2b.
Product: cI_434OR_cI434_EGFP on pSB3K3

(In principle, this plasmid potentially has a bistable property. That's our minimum goal.)

Step 4. PCR gene lacIq, assemble it into the product of Step 3.
Product: lacIq_cI_434OR_cI434_EGFP on pSB3K3

(This forms a complete switch plasmid. This is our intermediate goal.)

While Step 1-4 is on its way, we will go in parallel to build up the double repressor plasmid:

Step 1'. PCR cIind-, assemble it into the double repressor plasmid (pSB1A3 from I13521, Amp+).
Product: cIind- on pSB1A3

Step 2'. PCR any one of the double repressor promoter (noted as pDR ), assemble it into the plasmid obtained above.
Product: pDR_cIind- on pSB1A3

(Products of Step 4 and Step 2' form a complete switch system, at least in theory. This is our advanced goal. It would be a miracle if we could do more than that in one week.)

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* where we are
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As till tonight, we have done the following:

* As for PCR, we have successfully obtained the PCR product of 434OR, cI(both SDA & SDC), cI434(both SDcro & SDC) and EGFP(SDY) . They are ready for digestion (some are already digested).
That's all we need for the minimum goal.

* However, the PCR of lacIq and cIind- failed (for twice, once using LA taq, the other case using Ex taq).
The template for lacIq is the genome of some school of bacterial, that for cIind- is pLC003. Both templates are provided by LCB. I suspect there is either a problem with the templates themselves, or with the amount of template I use in the PCR system.

* We have successfully digested pSB1A3 (from I13521) with EcoRI & PstI.

* However, the digestion of pSB3K3 (from I7100) with EcoRI & PstI is problematic . Some plasmids are cut twice (which is good), yet some plasmids are only cut once (which is bad).
CCY will mini-prep pSB3K3 again tonight, followed by a double digestion. Hopefully the digested plasmid will be available for ligation (Step 1a, 2a) tomorrow.

* We have digested the PCR product of 434OR & EGFP with EcoRI & PstI. They are ready for ligation with pSB3K3^^(EcoRI/PstI). Though pSB3K3^^ is problematic (and a good one won't be available till tomorrow morning), CCY will do the ligation tonight.

In short, we are still in Step 1a and 2a. Hopefully we can move to 1b and 2b by tomorrow night (only if the ligation tonight is successful).


Best,
Daizhuo

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I will write a report of this kind every one or two days & I urge all Switch team members to read it carefully.

It's important that EVERYONE KNOWS WHAT'S HAPPENING IN THE LAB.

Daizhuo