Difference between revisions of "Part:BBa K3081058"

Revision as of 08:47, 21 October 2019

pBAD-dCas9-J23119-sgRNA

In the CRISPR replication inteference system, a 20-bp sgRNA is designed to be complementary to OriC, the genome replication origin. We use arabinose promoter to drive the expression of dCas9, while sgRNA is under the control of a J23119 promoter. Seven different targeting sites for dCas9 is designed to test the effect on cell growth. Binding box nomenclature used here is the conventional name of binding box (e.g. "M") followed by a "+" or "-" which stands for the direction of sgRNA from 5' to 3'. For instance, M box with its bottom strand bound by dCas9 is thus called M+ box.

This composite part is the principal design of the inducible CRISPRri system, but the intended sgRNAs are not assembled into the backbone, so this part contains a relatively meaningless sequence. Two BsaI sites flanking the meaningless sequence allow for easy assembly of intended sgRNA sequence by Golden Gate method. Length (412bp) of this meaningless sequence is far greater than that of normal sgRNAs (<20bp), facilitating easy detection of successfully assembled Golden Gate product when the colony PCR fragments are loaded onto gel.   After Golden Gate assembly, the ‘sgRNA’ sequence will be replaced by our carefully selected sequences (like M+, BBa_K3081107; R1+, BBa_K3081108; etc.). These newly generated pBAD-dCAS9-J23119-M+/R1/… parts each contains a meaningful sgRNA which guides the dCas9 protein to different boxes on E.coli genome replication initiation region, OriC, thus blocking the binding of relevant proteins essential for replication initiation to their boxes. Arrest and inhibition of genome replication initiation is achieved in this way.

Experiment

To precisely record the bacteria growth under stable conditions, a microfluidic chip is developed to adapt to observed features of bacteria (Figure 2C). All repeat groups are under flow of the same culture to ensure that the experiment results will not be affected by irrelevant external conditions. We have pointed out that interference of genome replication initiation would result in longer cell cycle and cell number doubling time. Here we take a 90 um * 90 um microscopic view each repeat group for cell counting every half an hour. It turns out that CRISPRri targeted to different boxes on OriC results in variant levels of cell doubling time extension, even though intervals between these boxes are only tens of base pairs. This is consistent with our expectations based on literatures, that functions and essence of different DNA boxes on the OriC and their contributions to genome replication vary a lot. Combined with known mechanism in DNA replication initiation, it is found out that our results accord with the DnaA binding affinity reported previously. High DnaA affinity boxes, like R1 and R3, were shown to have severe inhibition effect when targeted by dCas9. For typical low affinity box, like M box, the effect of CRISPRri is much milder. The only exception is R4, which was reported to be a high-affinity box but shows slight effect on cell growth.. Other boxes that are not considered to be DnaA binding boxes, like MR13 and IHF—they are, in fact, binding boxes for proteins other than DnaA—have mild effects. Moreover, we found another box which shows no inhibition ability on cell growth and the box happens to locate at the linker sequence of two DnaA binding boxes (M and R2) and is never reported to have specific biological functions before. These phenomena conform to our knowledge about DNA replication initiation that cooperative binding of DnaA is the rate-determining step.

Image:https://2019.igem.org/wiki/images/c/c2/T--Peking--polyAgif.gif%7CpolyA M+

R1+ R3+

R4- MR13

IHF M-R2+

Microscopic GIFs of bacteria transformed with CRISPRri system targeted to different boxes from microfluidic system. Transformed Top 10 strain is transferred to M9 medium in the ratio of 1:10 after overnight cultivation in LB medium. About 2 hours after transferring, bacteria in its log phase is precipitated by 5000-rpm centrifuging for 4 min and is re-suspended by M9 medium arabinose. Re-suspended bacteria are injected into the chip and observed and recorded continuously for 10 hours, under constant flow of 1 mL/16 hours.

Sequence and Features