Is the PACYCDUET vector compatible with the PRT28B vector?

Ask the expert: I am now doing the co-expression of four genes, using the dual expression vectors Pacycduet-1 and Petduet-1, but the activity is not as high as expected. I have encountered a series of problems, and I hope you can provide valuable experience and suggestions:

1) How to induce IPTG? My current method is as follows: activate overnight, transfer 100ml LB medium, incubate to OD=, add final concentration of IPTG induction, and induce at 25 degrees for about 5h.

Ultrasonic crushing, crude enzyme solution was obtained, and substrate was added and incubated for 24 hours. Is there a problem with that? Has anyone ever done a similar experiment?

Want some advice?

2) The protein I want is a soluble protein in bacteria, and purification is not practical because my enzyme requires four genes. So use a bacterial breaker. I collect the bacteria, ultrasonic crushing, with a power of 300w, 3s ultrasonic, 3s gap, 10-20 minutes.

Is there a problem with crushing like this? The general advice is that it is enough for the bacteria to become clear, but it is better to be slightly transparent, or to become completely transparent. Transparency in the literature has not been understood???

We hope you can provide valuable experience and advice.

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Clone Prawn 3rd Floor 2012-12-06 11:30:35

At least run an electrophoresis to see if all four of your proteins are expressed.

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susizheng 2nd floor 2012-12-04 14:39:09

The most in vitro enzyme catalysis does not need co-expression, or four genes, after separate expression and purification, and then put the enzyme together to catalyze?

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Yan Chunqiu 7th Floor 2012-12-06 13:00:19

The most in vitro enzyme catalysis does not need co-expression, or four genes, after separate expression and purification, and then put the enzyme together to catalyze?

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qingtingzhe 9th floor 2012-12-08 18:31:27

7th Floor: originally posted by Yan Chunqiu at 2012-12-06 13:00:19

The most in vitro enzyme catalysis does not need co-expression, or four genes, after separate expression and purification, and then put the enzyme together to catalyze?

The substrate is a macromolecular substance that cannot enter the cell, so it should be broken with crude enzyme solution. Some proteins are fragile and inactivated during purification, especially when it is difficult to ensure that four proteins are active at the same time. So it can't be purified! Thank you for participating.

Compatible, but try to cultivate a strain first to prevent genetic mutations.

The recombinant plasmid is inserted into the exogenous DNA at multiple cloning sites, and the upstream and downstream of the target gene are enzyme cleavage sites, and do not contain the start code and stop code. The target gene you design should contain an enzyme cleavage site, a complete protein gene (including the start code and the stop code), and the final target gene should be a protein gene sandwiched between two cleavage sites.

Of course, to add, responsible for how to form a ring structure.