Genetic Engineering

(BSM 3202)

WEEK III B

Learning Outcomes

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To be able to describe the natural occurrence of DNA transformation (C2) and the human applications of the process (C4)

CONJUGATION

Episome

• A plasmid that can integrate itself into the bacterial chromosome by genetic recombination.
• It carries a tra and a trb locus, which together are about 33 kb long and consist of about 40 genes.
• The tra locus includes the pilin gene and regulatory genes, which together form pili on the cell surface, polymeric proteins that can attach themselves to the surface of F-negative bacteria and initiate the conjugation.
• The pili themselves do not seem to be the structures through which the actual exchange of DNA takes place; rather, some proteins coded in the tra or trb loci seem to open a channel between the bacteria.
• Relaxosome - consists of proteins TraI, TraY, TraM, and the integrated host factor, IHF

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• The transferred, or T-strand, is unwound from the duplex and transferred into the recipient bacterium in a 5'-terminus to 3'-terminus direction. The remaining strand is replicated, either independent of conjugative action (vegetative replication, beginning at the oriV) or in concert with conjugation (conjugative replication similar to the rolling circle replication of lambda phage).

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• If the F-plasmid becomes integrated into the host genome, donor chromosomal DNA may be transferred along with plasmid DNA. The certain amount of chromosomal DNA that is transferred depends on how long the bacteria remain in contact; for common laboratory strains of E. coli the transfer of the entire bacterial chromosome takes about 100 minutes. The transferred DNA can be integrated into the recipient genome via recombination.

Hfr (high-frequency recombinant)

Conjugation occurred…but still “F^-”?

Transfer of genetic information in bacteria

BACTERIAL TRANSFORMASOME

BACTERIAL TRANSFORMASOME

THE ECLIPSE PHASE

• In theory, once a cell has taken up DNA -- containing a specific marker -- from the medium then it should be possible to isolate total DNA immediately from the newly transformed cells and then use that DNA in a second transformation -- selecting for the same marker.
• However, in naturally transformed B. subtilis cells, this is not possible -- no successful transformants will be found.
• They will only be found if one waits a period of time before isolating total DNA to carry out the second transformation.
• During transformation of B. subtilis, DNA from the medium is taken up as ssDNA molecules -- but B. subtilis cannot be transformed with ssDNA. So, until the ssDNA is converted into dsDNA as a result of recombination with the host chromosome, it will not be possible to obtain any transformants for the selected marker.
• The eclipse period is the time required to convert ssDNA into a stable dsDNA form.

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Interesting read

• Natural Competence and the Evolution of DNA Uptake Specificity, Joshua Chang Mell and Rosemary J. Redfield. J. Bacteriol. April 2014 vol. 196 no. 8 1471-1483. doi:10.1128/JB.01293-13

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• Natural competence for transformation. Blokesch M¹. Curr Biol. 2016 Nov 7;26(21):R1126-R1130. doi: 10.1016/j.cub.2016.08.058.

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Figure 21.2𝐵.121.2B.1: Bacteriophage

This transmission electron micrograph shows bacteriophages attached to a bacterial cell.