I am working with an engineered (Fusion) gene. It was provided in the cloning vector pUC57 by the company after synthesis i.e., a very high copy number vector. Gene is toxic for E. coli as it contains some part of ColE3, so I’m trying to clone it in Pichia pastoris. The expression vector we are using is pPICZαA. Our gene already has the kex2 signal cleavage site and His-tag for purification, therefore, we want to use the XhoI restriction enzyme so that it can remove the kex2 signal cleavage site already present in the vector. Now, as the MCS of pPICZαA has 2 XhoI sites, we are doing sequential digestion firstly digesting it with KpnI (KpnI site is present just beside XhoI’s site, so it disrupts XhoI site), then with XhoI. Likewise, the company has given restriction sites for XhoI and KpnI for separating Insert from the pUC57.
pUC57-Amp: 2,700bp
Insert: 2,328bp
pPICZαA: 3,600bp
XhoI- ThermoFisher FD Enzyme
KpnI- ThermoFisher FD Enzyme
T4Ligase: Thermo
Problems:1) I have tried to transform it in T10 and XL1Blue competent cells, but transformation only occurs in XL1Blue and its growth is very slow on both agar and broth (Both competent cells are fine as I’have tried transformation with another gene).2)Cells do not revive from glycerol stock (15% glycerol). 3)After sequential digestion of pPICZαA with KpnI, then with XhoI and double digestion of insert with KpnI & XhoI, I have tried ligation in the following conditions: a)In the ratio 3:1, 5:1, and 7:1.b)At room temperature for 2 hours, at 16 ºC for 4 hours, and at 4ºC for 16 hours. But I’m getting false colonies (10-12 colonies). So what could be the reason?
It is known that lac promoter is leaking and some expression of a sequence under it still occurs even without direct supplying of lactose in a cultural media. There is a possibility that a toxic insert forms an ORF at pUC57 and is expressed mildly. In that case, changing of cloning vector would help to avoid promoter leaking.
Igor Oscorbin On the first hand I had the same doubt, but the gene is present in the opposite direction of the lac promoter.
Hi Jyoti,
Are you sure that KpnI digestion disrupts the second XhoI? I checked the sequence provided by Snapgene and it seems that after digestion with KpnI the XhoI in the MCS is still there... (See screenshot attached).
The ratio for that ligation should be reduced. Your vector and gene of interest have more or less the same size, a ratio of 1:1 using 50ng of each should be more than enough. But first, fix the restriction enzymes issue.
Anyhow, the result of the ligation you performed should render empty pPICZαA vectors, I cannot think of a reason for them to grow slow. E.coli not growing after streak out from glycerol is weird... I normally use a higher concentration 30-40% of glycerol.
Also, I have worked with pUC57 plasmids with synthesized genes in it and the copy number of this plasmid is not very high, average yield for a mini prep is around 50-100ng/ul but not much more.
Good luck.
Xi Jiang It exactly does not disrupt the second XhoI site, but as we know that these restriction enzymes are endonuclease so they at least need two extra nucleotides to act on the restriction site.
And the second point is, that the slow growth is seen after I'm trying to transform the insert with the pUC57 vector that I'have got from the company, not with pPICZαA.
We have other customized genes in pUC57 and their average yield is 300-400ng/ul.
Yes, endonuclease require extra nucleotides to cut efficiently but still it can digest the fragment even if there are no extra nt. In any case, there are some extra nt in the antisense strand as you the cutting site of KpnI leaves cohesive ends. (XhoI site in bold)
5'-______CTCGAGCC-3'
3'-CATGGAGCTCGG-5'
I still think the problem of the false positive colonies after ligation is due to XhoI site being digested after KpnI and false positive colonies come from recircularization after joining of XhoI cohesive ends. You may send for sequencing a couple of colonies for sequencing using primers close to the MCS to see what happened and get a conclusion. Anyway, ligation ratios should be more similarn(1:1) as the size of fragments are similar.
Xi Jiang Yes you were right, the problem of false colonies after ligation was due to XhoI site being digested after KpnI. I tried another enzyme NotI instead of KpnI and ligated at ratios 1:1 and 1:2, ligation was succesful.
Thanks for the suggestion.
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