Transforming E. Coli cells with a custom hTERT plasmid
Okay I'll begin by explaining as best I can what exactly I'm trying to do. I'm trying to transform E. Coli cells. I want the cells to uptake a custom
human telomerase reverse transcriptase (hTERT) plasmid. Now I say a custom plasmid because in addition to containing all the genes necessary to make
hTERT I also want this plasmid to attach a protein transduction domain (PTD) onto hTERT. In theory the PTD should allow the hTERT to enter cells in
vivo, that is to actually pass through the cell membrane and into the cytosol where normally it would not be able to. Once I have successfully gotten
a culture of E. Coli to produce the modified hTERT plasmid I intend to cultivate them for quite a while. I want to accomplish this before I move on to
the next part of the experiment. I describe the next part in the paragraph below but be aware that I haven't though the second part out as well as the
first.
In the next part of this little experiment I intend to lyse open the hTERT producing bacteria and then filter out everything but the hTERT as best I
can. At this point I want to do a little study on feeder mice. Basically I'll have a test and control group. The test group get regular injections of
the filtered lysate which should contain hTERT and the control will get saline injections. Ideally I'd have a second control group which receives a
lysate injection containing hTERT without the PTD add on but I'll get to why I'm probably not going to do that later. Anyways I'll take a blood sample
from each group prior to injecting them with anything. I intend to measure hTERT presence/activity in the cells of each group using one or two
methods. In the first method I'll filter each blood sample so that I separate the red blood cells from the plasma which is supposedly easy to do. I'll
then lyse open the red blood cells. I'll filter this lysate as best I can so that only hTERT is present (which it shouldn't be or in only very low
amounts since it normally isn't present in somatic mice cells). Then I'll measure hTERT presence using electrophoresis. I'll then take regular blood
samples of each group after I start hTERT injections and again measure hTERT presence via electrophoresis.
I realize that this is a potentially flawed method of measurement since one would expect the test group to have significant amounts of hTERT in their
blood stream from the injections. I'm hoping I can somehow figure out some sort of solution to this problem by either some clever timing (ie waiting x
amount of time after the hTERT injections to take blood samples) or maybe also looking at how much hTERT is present in the plasma as well or it's rate
of change over time. Again I haven't thought about the second part of this experiment so much. I'm more concerned about getting a stable line of hTERT
producing bacteria.
I mentioned I might use a second method to measure hTERT. Basically I would need to find some method of measuring telomere length that is easy, cheap,
and can be done at home. In theory if the mice have hTERT present in their cells then it should elongate their telomeres, or at least slow down the
rate of degradation.
So anyways that is the experiment I'd like to run. Now the reason I posted here is because I'm looking for both general advice as well as answers to
specific problems. At the moment I'm only concerned with successfully transforming the bacterial cells. I imagine I'll have to cultivate quite a lot
before I can move on to testing with mice so there's no point in worrying about that now. Now obviously if I've overlooked something (and there is a
decent chance I have) please point that out but here are the specific problems I'm facing and would like help with.
The first problem I have, and the most challenging, is getting the materials I need to do this. The actual hardware involved (dry bath, micropipette,
etc) is easy to get and relatively cheap. However I'm not sure where to get the plasmid I need made. Most places aren't going to sell to individuals.
I've found one website that seems to (origene.com) but they are very expensive (>2k$) and seem to only make custom plasmids for institutions
(though they do seem to sell 'normal' hTERT plasmids to individuals). PTDs are fairly short sequences usually around 30 base pairs so hopefully it
shouldn't be too expensive to tack one on to an existing hTERT plasmid.
Secondly I'm not entirely sure what sort of PTD to use. This page here gives a great overview of them:
http://dissertations.ub.rug.nl/FILES/faculties/science/2006/...
At the moment I'm leaning towards using PTD-4 as described in that document but if someone knows something about them and can suggest a better one
that would be amazing.
Thirdly it seems that most companies ship plasmids on dried filter paper. Every time I've worked with plasmids in the past they have already been in
solution. I've read online that all you have to do is cut out the area of the filter paper with the plasmid in it, stick the paper into an eppendorf
tube with some TE buffer in it, then swirl it with a pipette tip. I just want to make sure though since the plasmid is probably going to cost a lot
and I don't want to ruin it.
The last problem I have isn't really a problem. I just want someone to make sure I'm using the right procedure for heat shocking the cells into
uptaking the plasmid. It's been over a year since I've done it myself and just wanted to make sure I hadn't overlooked something. I plan on using the
protocol outlined here:
http://www.stanford.edu/~teruel1/Protocols/pdf/Transformatio...
So if anyone could help me with these specific problems as well as giving general advice I would be very grateful (even a "yes that sounds right" is a
great confidence booster).
[Edited on 28-12-2011 by 1101]
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