Part:BBa_K1433016:Experience
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Applications of BBa_K1433016
Validation of Chromosome Recombination with λ-Red Technique
Growth curves under L-arabinose gradients induction
In order to test if the basic genome-editing part---λ-Red system works, we designed several different knock-in site pairs on E.coli chromosome and utilize antibiotic resistance gene to target them.
Firstly, from a large number of paper collection, we summarized aλ-Red protocol which tells that recombination bacteria cell preparation requires L-arabinose induction, and L-arabinose appears to inhibit cell growth due to cytotoxicity ofλ-Red proteins. Therefore, as to learn about cell growth circumstances under L-arabinose induction, and test ifλ-Red proteins get expressed on some level, we made a growth curve of gradient arabinose induced cell growth as follows. Through the curve, we can easily find that with the increase of arabinose induced concentration, cell grows slower and slower, implying the inhibition effect got strengthened.
Homologous sites choose
hen we sought to find cause our “socket.coli” needs a target site to be explored as a knock-in platform, which means all of our chromosome circuits should be inserted into such sites. Confronted with as long as 4.6Mbp E.coli genome, several criteria of our homologous site pair are supposed to be considered. Firstly ,it is not less than 40bp and more than 70bp, in that too short a site is not sufficient to implementλ-Red-mediated recombination, and too long is not efficient to add these homology sites to each ends of targeting fragments. Secondly, since the targeting fragment is modified via PCR, the secondary structure of both site pairs shouldn’t be too awful and most desirably meet the criteria of designing PCR primers. In addition, both sites are not on essential genes. Last but not the least, the region flanked by those two sites should be as suited as possible to insert 1~3 kb targeting fragment, for all of our targeting fragments vary from 1kb to 3kb.
Corresponding to the criteria mentioned before, we chose two different homologous site pairs both of which lies on the lac operon gene, which doesn’t affect the viability of bacteria cells at all. As to deal with targeting fragments of different length, two site pairs are chosen to be the socket target site. The two pairs are named respectively as HL/HR and LacHL/LacHR. Of the two, HL/HR is selected as a target for antibiotic resistant gene knock-in validation, and LacHL/LacHR is mainly used to knock in the chromosome circuit part.
Recombination towards HL/HR site pair
Through recombination towards HL/HR site pair , we demonstrate the basic knock-in function ofλ-Red system. The targeting fragments include two kinds of antibiotic resistance genes, kanamycin-resistant gene and tetracycline-resistant gene. Both antibiotic resistant gene are added HL/HR sites by two rounds of PCR.
The positive recombinational cells grow in the corresponding antibiotic LB plate followed by electrically mediated targeting fragment introduction. Using HL/HR lateral detect primer pairs, negative control gets a fragment of the length of 1767bp; on the contrary,if targeting fragment recombines towards the chromosome successfully, a band as long as 1308 bp will appear, which from the electrophoresis gel image, expected results shows up.<p>
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