transformation of polyploid industrial Saccharomyces - (Oct/09/2008 )

I have tried to transform industrial S. cerevisiae with pRY255 integrating vector (17.9Kb) with amylase gene (1400bp). In this process, i first amplified amylase gene, then digested both pRY255 and amy by Sal I RE, after that i ligate both pRY255 and amy using fermentas ligation kit and again digested the ligation mix with Kpn I, also KpnI site was found in amy gene too, that's why i try to partially digest it.

KpnI was used to linearlize the vector pRY255, then i tried to transform the mix in S. cerevisiae using Invitrogen kit. However, i didn't find any colonies.

can any one suggest what should be the correct molar ratio of amy:vector? or suggest the concentration of dna to be used.

please suggest what could be the remedy for this kind of work.

Thanks & regards

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Do things one by one dont' rush.

First get your ligation to work first . just do a ligation and transform and find those with insert.

THEN,

1. use something else other than KpnI because ur amy gene contains the KpnI site as well...
2. if u were to attempt partial .. try changing the duration of digestion but that alone might not help much . Partial digestion really depends on several factors such as the accessibility of the DNA region, u might always end up cutting the Amy gene first before getting to the other site.
3. Could you tell us more about your cloning strategy? Are you attempting a Homologous Recombination or some sort. More detail would be greatly appreciated to help us to help you.,

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Dear Hanming

Thanx for ur reply.

I am intrested in homologous recombination for cloning purpose. pRY255 vector has Ho gene which is also present in yeast chromosome and KpnI site is present there to help in recombination.

I would like to go through by linerizing the vector with KpnI, becoz i don't know other RE having a single site.

My cloning strategy is:

1) Isolation of vector and amy gene

2) Restriction digestion of vector and amy gene with same RE (SalI)

3) Ligation of vector and amy gene

4) Partial digestion of ligation mixture with KpnI (This is for linearizing the vector)

5) Transformation in competent Yeast cells, prepared with SC easy comp Transformation kit (Invitrogen)

6) Plating on selective plates

7) Incubation for 3-4 days

Kindly suggest where should i check for any flaws

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Everything is looking good. My concern is the direct linearization right after ligation .
The amount in ligation is pretty low but high enough to ensure transformation , however using the amount in ligation to do a restriction digest and then do a transformation and then hoping for a homologous recombination (HR) would be a tough one IMO.

I am not a pro in homologous recombination in particualr but what i know is its efficiency is not very great.
Transformation efficiency is also low and when u combine with the low efficiency of HR, u really can go buy a lottery if u got a confirmed colony of interest that way.

If you could tell me the mechanism of the homologous recombination you're doing i would greatly appreciate that.

But first thing first let's get just the ligation product before proceeding to the next step of linearization + HR shall we?

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you are right dear,

it is very difficult to get a perfect combination of all.

I wanted to know that, do u work on yeast?

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No, i work on bacteria . Therefore i only comment on the cloning strategy and ask about the homologous recombination thing that you're doing but i do however remember having to take my final genetics test with all sort of Yeast question lol . was a fun one calculating recombination frequency and all that.

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