Okay need feedback on my idea - (Jan/24/2010 )
Hey everyone. I'm a grad student.
My prof wants an independent research idea (no biggie. a simple research) and wants me to work on it for atleast 45 hrs this sem to c if im gud enough.
I'm havin a lot of ideas although a lot em get ruled out cz theyre either nt ideal considering the time constraint or theyre too elaborate. So i was thinkin bout designing a simple recombinant experiment. So heres what i came up with: (im yet to decide on the vectors, so plz bear with me)
1. Two vectors, one with a GFP protein and another a pET vector.
2. Digest them with same restriction enzymes and ligate the GFP to the pET vector.
3. Transform the recombinant vector into BL21(DE3) strain.
4. BL21(DE3) has T7 RNA polymerase in its genome under the control of a Lac operon.
5. Induce the transformed BL21(DE3) with IPTG. Monitor the expression.
Good enough considering the short time?? I know it's not novel enough but that I was wondering if I could use some sort of method of detection to light up the GFP atleast for my curiosity (will be cool to see em light up)!! Plz i need your feedback guys and if its crappy plz say so. Also, are those monitoring GFP in E.coli cells normally done? I mean plz do correct me if I'm wrong. Thanks. Looking forward for your feedback.
Not a bad idea all...to start with. Dont worry, even if you go wrong. Only thing you should know where and why you are going wrong. I will mention my suggestions as per no. of points that you have mentioned.
1. Ya, you can get such two vectors.
2. Everytime you have to confirm digestion of insert and vector. Vector should be SAP treated, to prevent selfligation.
3. Thumb rule is to transform, 2 tubes of comp cells, one with null vector i.e. without insert (cut and SAP treated) and other with insert. You should get more colonies in vector+insert. Ideally you should get no colonies in cut vector (which is SAP treated) but generally you get few colonies.
4. Yeah, should be the case.
5. Considering, 4 is true. You generally induce with 0.1-1.0 mM IPTG, you can use 0.1 mM. Before that, you have to preapare a starter culture of the clone and then inoculate it into 100ml shaker flask at 37 degrees.
Once the OD reaches 0.6-0.7, then induce with 0.1 mM IPTG and change the temperature to 25-30 degrees.
6. You can keep about 1ml of induced sample. Remove samples after one hour. Check on SDS PAGE whether you are getting a induction (that is a increase in protein conc. at desired size).
Hope I have not stuffed you with too much of information.
Let me know if you need any clarification.
Best of Luck
Thanks for the feedback. The problem is i have not decided on the actual vectors to be used. I've a general question. I know I stated am example of GFP but are there vectors with similar proteins which can be expressed in E.coli? (i mean apart frm the regular lac, MCS and antibiotic resistance which are present in almost all vectors) if so, do the company (novagen, invitrogen etc) websites have them?? i hope ur getting my question. And I've to check the compatibility of these vectors before i design the experiment right ? in terms of restriction sites etc. I hope my questions are not too simple minded. looking forward to your reply. Thanks again for your support.
There are vectors available with clonetech which have GFP gene + amp resistance.
Essentially, you need not reclone them into pET vector, you can directly transform it. But since you want to try this out, you can do it. You have to find two pairs of enzymes, which do not cut the GFP gene, but cut the vector. You can refer the manuals of both GFP-based vector and the pET vector for this.
Hey sorry for the delayed thanks
I submitted my outline 3 days back to my advisor and she wanted to know how I would insure that the GFP coding sequence will be in frame with the T7 promoter to express the GFP protein? So, I was thinking along the lines of a western blot (using an Anti-GFP protein). Is this right? Or are there expression assays which you can do to see if the protein is expressed or not?? My doubt is, only if the protein is in frame with the T7 promoter can it be expressed right?? So, why not assay the expression?? Please let me know your thoughts. Thanks
if you use the ndeI site to clone in your gfp, it will be in frame with the T7 promoter. the ndeI site is catatg, and atg is the start methionine. all pET vectors have this.
also, it will be clear if the protein is in frame or not when you grow and induce the cells. the cell pellet at harvest will be neon green.
The promoter does not have a frame -- it is a DNA sequence recognized by RNA polymerase -- therefore it need not (indeed, can not) be "in frame" with anything.
Now, the fusion tag -- that's a different story...