Hi everyone, it is my turn to share again =) Remember the first posting on the construction of the virus? Well, that is just a brief outline of what I have been doing for the past 6 weeks and will continue to be doing for the next few weeks.
*(Later I will share with you why it takes such a long time) ==> remember this one of the big question for this post, if you can answer this, then you did a *good job in understanding this entry =) heex. Those who cannot..(no such thing k) lols.jk. can ask me =)
Basically, because I am attached to a research lab, I planned to share more of some of the techniques the labs are using nowadays for molecular biology research work and some cell culturing techniques in the later posts.
I will break down the procedures into a few separate postings so that more detailed learning of each of the techniques can be shared.
For the procedure in the construction of virus it involves two MAJOR components.
1. Cloning
2. Packaging into the virus (I haven reach here! so this will be shared next next time k, because I foresee first step will take very long==> why? Find out by continue reading..)
1. Cloning
Cloning in this context refers to isolating a defined DNA sequence and obtaining multiple copies of it. Cloning is use to amplify (by using living cells such as bacteria E.coli) the specific DNA sequence containing the (gene of interest)*secret.
E.coli is often used because of the easy manipulation and it can produce high copies of plasmid.
(the gene of interest)*
E.g. Gene A ==> has to be cloned==> packaged into the virus==> get virus A==> introduce gene into mammalian cells
E.g. Gene B==> has to be cloned==> packaged into the virus==> get virus A==> introduce gene into mammalian cells
Each (gene of interest) has to be clone separately because each gene size is very big: up to 2 to 2.5kb.The longer the sequence of the gene the easier/ higher possibility of getting mutation in the gene when we are doing cloning. This is because during the procedure of cloning, it involves a lot of manipulation of the gene, especially PCR which can introduce single bp mutations. Even if very high fidelity (means accuracy of the copy to the source) PCR mix is used, the PCR process is not 100% perfect.
Bacterial cells containing the gene of interest is provided by the manufacturer in a very interesting form. It is in a small transparent glass bottle (cylinder shape) which has ¼ filled agar inside, and one very big white colony is seen sitting on the agar. (The bottle is only about 4cm in height and 1.5cm in diameter). The manufacturer provided it in the bacteria form on agar for easy usage, because we can just pick and use the bacteria.
Cloning can be further broken down into 4 main parts- this is called the “Cloning Strategy”
1(a) Isolation of DNA insert
1(b) Ligation
1(c) Transformation
1(d) Screening (Blue-white) / Selection strategy (antibiotics)
According to the procedures shared in post 1, this is a summary map of the procedures. (Construction of virus: cloning and packaging into the virus).
You CAN click on the picture
Picture taken from http://www.invitrogen.com
I am currently still in the green box, which means I have gone through these steps:
1(a) Isolation of DNA insert ==> Original (gene of interest) purchased from manufacturer, that comes in the form of plasmid contained in bacteria cells.
1(b) Ligation ==> of the (gene of interest) into Vector Backbone containing 7nTR (Facilitate later steps)==> to get a recombinant plasmid.
1(c) Transformation==> of recombinant plasmid into first type of E.coli cells (lets call it 5a)
1(d) Selection strategy ==> of the transformed cells (5a) (using amphiciliin antibiotic)==> then pick a one colony randomly *clue and culture it in broth *(15hours) ==> extract the recombinant plasmidè restriction digestion to cut out gene of interest and run on gel to confirm presence of gene of interest ==> and send for sequencing (using the fluorescence labeled ddNTP) to check for any mutation of the (gene of interest) IN THE recombinant plasmid ==> if there is mutation of the bp (seen in sequencing results) ==> go back to the plate and *re pick the colony, re grow them and sequence again. (thats partly why my lab people always say "pray hard!"==> cos its like kinda 'chance' thing even when optimal conditions are provided.)
Cloning process can take up to 1month if every goes well, but more than 1 month if we keep re picking the colonies. Usually, we will number the colonies on the agar plate, so that we know if that colony has already been picked before.
Sequencing process is done by sending it to an external company which does sequencing, E.g. The company 1st base. We have to provide the samples (picked from colony) and primers specific to the gene we want to sequence. Usually sequencing results using the fluorescent ddNTP method starts to become less sensitive when it reaches the later part (~700bp), therefore one primer for every 700bp we want to sequence should be provided). Sequencing *results usually takes about 3 to 4 days to arrive and usually a soft and a hard copy will be provided. The soft copy shows both the sequence in e.g. “actgactg form” and the electropherogram, while the hard copy shows us the electropherogram results with different coloured peaks but the different bases (a, t, g, c) only.
In the green box, it shows a recombinant plasmid which I am supposed to get to proceed to transformation of second type of E.coli cells (lets call it 10a) ==> shown in the picture named as “competent E.coli cells”.
Steps for :
Isolation of DNA insert
Therefore once I received the bacteria cells (gene of interest inside) ==> plate them on LB +amp plate to get single colony==> pick one colony==> grow in broth (15hours) ==>extract the DNA è PCR to amplify the gene of interest and introduce restriction sites è restriction digestion ==>
Ligation, transformation and selection
Ligation==> transformation ==> selection==> pick colonies ==> grow them in broth (15hours) ==> extract the DNA ==> restriction digestion to cut out the insert (confirm insert is inside recombinant plasmid) ==> sequencing (using the fluorescence labeled ddNTP)
Most of the techniques are just recapped from the molecular genetics and molecular Biology and culturing of bacteria from basic microbiology.
One thing which I found very interesting is this technique: Cloning of PCR product, I ask the other students and a lot people never heard of this before in school! so it’s a good thing to know =)
PCR refers to polymerase chain reaction, it is a process which allows the amplification of any region or even very complex genomes in just a few hours. (for details refer to link)
http://bcs.whfreeman.com/lodish6e/default.asp?s=&n=&i=&v=&o=&ns=0&uid=0&rau=0
go to this website, click on animation and then PCR
Cloning of PCR product
PCR product can be directly used for coloning? How?
A restriction enzyme recognition site can be added to the 5’ end of the oligonucleotide primers used for the PCR reaction. These sequences will also be into corporated into the amplified PCR product as the PCR cycles continue, it can later be digested to produce the specific sticky or blunt ends for ligation. The primer will bind to the complementary part of the DNA template, but the restriction enzyme recognition site does not match the template. However, since the direction of synthesis occurs in the 5’ to 3’ direction and specificity depends mainly on the 3’ end of the primer, the DNA still can be amplified efficiently and the product will contain the restriction sites at its end. Extra bases are also eventually and normally added to the 5’ end of the restriction site to ensure that the restriction enzymes functions efficiently.
Taken from PCR by (1997) second edition from C.R. Newton & A.Graham
Hope you learnt new things! =) Take care!
Jean Leong
TG02
0607991G
Reference
1. C.R. Newton & A.Graham.(1997) PCR (2nd edition). BIOS Scientific Publishers Ltd, USA.
