Bacterial mutants are constructed through homogeneity recombination of suicidal protons

of the nature of suicide granules: generally used to
gene
mutation
. Cloning the mutant's destination gene to a suicide-type granule vector and entering it into the host through bonding, etc., cannot be replicated because there is no replication protein (e.g. Pi protein, etc.) required to replicate gene initiating in the host bacteria, and the suicide-type granule carrier carries under the influence of external selective pressure. The mutant gene is recombined with the wild type on the host chromosome, resulting in a mutant strain with a mutation, while the prosurge vector itself disappears from the bacteria along with the original wild type gene on the chromosome due to its suicidal properties. As for a recombinant mutation, it can be screened with the help of selected genes on the protons (e.g. sacB, sucrose sensitive).
integrated vector: cloning the gene of destination on the prosurloy vector and integrating it into the host chromosome is an effective way to overcome the instability of the prosurloy.
I. Regarding the carrier
suicide-like granule vector can be completely constructed with itself, as long as two points are guaranteed:
(i) suicide-like granules carrier can not be replicated in your host bacteria, that is to say, there can be no replicated replicated in the host bacteria on the vector.
the suicide vector cannot be replicated in your host bacteria, otherwise it will cause the construction of bacterial chromosomal mutants to fail. However, when building, be aware of the following:
1, in the preparation of prosaru carriers and
DNA
insertion fragments: 2ug 5kb size line DNA contains approximately 1.4poml/L5' end phosphoric acid. 5' Protruding end dephosphorusing requires CIP0.01u/pmolDNA end, flat end or depression dephosphorus dephosphorus requires CIP0.5u/pmol DNA end.
2, connecting prosurgery vectors and DNA insertion fragments:
(1) The first experiment can establish several different connection systems, such as the ratio of inserted fragments to vector molecules can be 3:1, 1:1, or 1:3.
(2) can try three temperature and time connections such as 4 degrees overnight; 15 degrees, 4-6 hours; 25 degrees, 1 hour.
3, bacteria
transformation
and screening:
(1) positive control: if it is
receptor cells
, -80 degrees stored after 5-6 weeks, the conversion rate is low. The conversion ability of receptor cells can be identified using a known standard closed-loop granules.
(2) negative control: with non-DNA transformers of the receptor bacteria paving, if there is a growth of bacteria, indicating that the concentration of
antibiotics
is not enough, or receptor pollution
(ii) screening markers
2, gardeners experience and recommendations
1, generally do not need to induce the expression of recombinant enzyme genes to carry out effective omogenous recombination. Unless your strain is recombined, this is another matter. Of course, in doing suicide-like particles is to consider the length of the exchange arm, the longer the arm mutant strain is easy to get, the less work before screening. In general, the two arms in the exchange guarantee about 1kb of the same source sequence, can improve its switching efficiency.
2, Q: If I want to build a mutant strain of a gene of Bacillus sprouts, (1) mutate the gene in-body
; the
sequence of nucleotides; (3) recombines the DNA fragment into the prosurages of an E. coli; (4) linearizes the recombinant prosurges; and (5) converts linearized granules into dead herb; Screening recombiners: Can the construction of the dead herb mutation be completed through the above 6 steps through the same source recombination? (Gardener qzh21sohu)
A: (1) After the suicide prosurges are constructed, they do not need to be linearized and can be transferred directly to the subject bacteria.
(2) you can get a positive clone in these 6 steps, but you can't filter because I didn't find a screening marker (such as antibiotic resistance) in your step, which is usually in the middle of the gene to be mutated. But if you're not infested with a gene, but just a bit of a mutation, you can't put the screening marker in the middle of the mutant gene, but you should follow it, adding the corresponding antly source sequence (around 1kb) to both ends of the mutant gene and the screening marker. But there is also a question, is you want to mutant gene is singshun anti-sub or multi-shun anti-sub, if it is more shunned anti-sub, then it is in the position? Because you have to consider not affecting the transcription and translation of other genes. Please check it carefully.
(3) different bacteria use different suicide prosultons, which are named because they cannot be replicated in the subject bacteria. But try to see if this prosulte can replicate in Bacillus spores.
(4) to check the relevant literature on the Internet, there must be experimental steps. That's what we do.
3, not any bacteria can be made into a high-efficiency state of feeling. If the suicide vector does not have a mob bit, what method should be imported (combined) into the vector bacteria? Gardener Wuji suggests a method:
the phage
transducting. My sister-in-law imported the prosaic particles into Salmonella using Bacteriophage P22.