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0:04
So the big question is,
how do the de novo proteins function?
Two alternatives.
It would seem logical
that the de novo protein catalyzes
the same reaction
as the deleted enzyme.
That's seems totally reasonable, right?
However,
that's not necessarily the case.
It might also perform the rescue
by somehow increasing the expression
or enhancing activity of some
endogenous natural E. coli protein.
Hence, we have to consider
both possibilities,
the first simple one
and the second more complicated one.
So which is it?
And the answer is, yes, it's both.
We have examples of both cases.
Let me go through the second one first
and then I'll go through
the top one afterwards.
So enhancing the activity
or expression
of an endogenous E. coli protein.
0:51
On this next slide,
I'm introducing you to SynSerB,
and so that indicates
the synthetic protein Syn
that rescues the deletion of SerB, okay,
and SerB is the gene that codes
for phosphoserine phosphatase,
that's essential in the biosynthesis
of the amino acid serine,
and the key reaction
is shown on the top right.
It's a phosphatase
that removes out phosphate group
from phosphoserine to yield
the essential amino acids serine.
If that gene encoding that enzyme
is deleted,
then the cells cannot grow
on minimal medium.
As you see in the panel
on the bottom left,
if we grow cells in minimal medium,
delta SerB cells,
these deletion cells, in minimal media,
if we put in the control gene
encoding the control protein LacZ,
nobody grows, dead, baseline, flat line.
However, if we put in the wild type,
the natural SerB,
things grow rather quickly
as you see in blue.
Alternatively,
if we put our synthetic protein in,
or more specifically, a synthetic gene
encoding our synthetic protein,
we get growth, not surprisingly,
it's not as good as natural wild type,
but yes, surprisingly,
it actually works.
This novel protein sustains life.
So how does it work?
We purify the protein
and we see that in vitro
the purified synthetic protein
does not catalyze the reaction
showed on the top right.
Perhaps there is something
in the cell lysates necessary.
Nope, the cell lysates
don't do it either.
So we're forced to the conclusion
shown in purple here
that the rescue mechanism
is not catalytic
but somehow regulatory.
But we don't know what it is.
We really have no idea where to look.
And so we did a rather involved
experiment here called RNA-seq,
RNA sequencing,
and we asked which genes in E. coli,
which RNA transcripts are enhanced,
are turned up by the presence
of our synthetic protein,
our artificial protein SynSerB.
