We have successfully demonstrated the assembly of 5 BioBytes within one of our microfluidic chips. The construct was made up of the anchor, 5 BioBytes (2 genes that alternated), and the terminator. Each BioByte chamber held 4.2 uL of their BioByte along with ligase and ligase buffer. This is a very small amount compared with the volumes used in the off-chip protocol. The beads were left in each chamber for 15 minutes. The total time was less than 2 hours and was executed at room temperature. After digesting the construct off of the bead with the USER enzyme, the construct was ran on a gel. The gel shows 5 very dim but distinct bands (which is expected with the low reagent volumes). The size of the construct implied by the bands indicate that the assembly process was a success. Repeated experiments verified these results.

The XY stage in the robot was built by a few undergraduates here at the U of A as their capstone design project. This XY stage is what moved the magnet around which controlled where the beads went on the microfluidic chip. Since then, this XY stage has been incorporated into another machine meant to combine many lab-on-a-chip functions into one device including PCR and CE for sequencing. That is why the machine is so big; the internal laser and camera take up a lot of space. The software can be controlled from a Mac or PC. Since the chip's channels were curved, it was somewhat cumbersome to attain the XY positions that the magnet needed to go. But once the positions were known, the rest was easy.

Q. What stops the DNA in the outer wells from diffusing into the washing chamber and contaminating all of the other chambers?

A. We set up Laplace flow originating from the central washing chamber.

Q. What is Laplace flow?

A. Laplace flow is the term used for the flow of liquid caused by differences in pressure. So by filling the central washing chamber to a height slightly higher than the outer chambers, a slow and steady flow against the concentration gradient of the DNA was accomplished.

Q. How do you stop the chamber contents from evaporating?

A. We normally cap the chambers with a small volume of oil. Without the oil, the small reaction volumes can evaporate in less than 5 minutes.

Q. What are some of the physical characteristics of the microfluidic chip?

A. The chip is made of 2 glass layers. The micro-channels are etched onto the top of the bottom layer, and the chambers are part of the top layer. The channels are 45 micrometers by 100 micrometers. The chip itself is approximately 1 inch by 3 inches. Each outer chamber can hold 5 uL of liquid. The central washing chamber volume varied from chip to chip ranging from ~2 uL to 20 uL. Some chips had a closed central washing chamber, but were no longer used after unintentional Laplace flow was found to be largely uncontrollable. The chips are reusable after a quick wash.

Q. How do you fill the channels?

A. Once liquid touches the central washing chamber, capillary flow rapidly fills the channels. The outer chambers are then ready to be filled.

Q. Why were the channels made to curve like they do? Why are they not simply coming straight out of the washing chamber towards the outer chambers?

A. The first reason was to increase the length that the DNA had to travel if diffusion turned out to be a problem. Since there was limited space, curving the channels is all we could do to increase channel length. The second reason was so that we could visually confirm that Laplace flow was working by filling the outer chambers with orange dye, and the central washing chamber with blue dye. Shorter channel lengths would have made this hard to see.

Q. How do you know that the beads do not drag unwanted DNA around to contaminate other chambers?

A. In our tests, we've recovered the contents of the chambers to run them on a gel and see if any DNA from a different chamber has entered. No measurable amount of DNA was found to contaminate other chambers (including the wash chamber). This suggests that the Laplace flow from the washing chamber is enough to cleanse the beads of DNA that has not ligated.

Many of the procedures needed for BioByte assembly have been accomplished on a microfluidic chip in labs across the world. Although not used for BioByte assembly and not accomplished on the same chip, these on-chip protocols have shown that PCR, purification, digestion, electromagnetic bead manipulation, ligation, cell separation, and electroporation (transformation) can all be achieved on-chip. The future will bring many of these procedures together onto one chip allowing the researcher to choose their artificial plasmid on the computer, and upload the program to the microfluidic chip's microprocessor.

The diagrams above show what a Biofab-on-a-chip might look like. It would be a large scale integration of previously independent lab-on-a-chip procedures. Starting on the left of the first labeled diagram, BioBytes are PCR'd out of their plasmids and their sticky ends are created after the USER enzyme is pumped in. The solution needs to be PCR purified and is pumped through the wavy channels. The DNA does not make it through at first; the waste is made to flow into the waste chamber. Soon after, the purification buffers are pumped through allowing the BioBytes to collect in the BioByte chambers. Prior to this, magnetic beads were prepared by being dragged into the anchor chamber (either by an external magnet, or on-chip micro-electromagnets). When the BioBytes are ready in their chambers, the supply of beads is split into, for example, 4 portions. Each portion is transferred to a different BioByte chamber and attaches a BioByte to the anchor. After ligation is done, each portion of beads is led to another chamber (through the wash canal) where each bead portion attaches a second BioByte. The process continues until a certain amount of BioBytes is on each bead. Two bead groups are moved to independent MegaByte chambers where the USER enzyme cuts the constructs off of the bead. The beads are transferred back to there storage chamber while the construct is left free in the MegaByte chamber. The remaining bead portions that still have constructs attached are transferred to these freshly cut constructs and soon ligate. There are now two bead portions left, each having a construct. One of them is cut with USER, and the remaining bead portion is moved to that chamber where the constructs ligate. At last, this large BioByte construct is cut with USER and is ready for electroporation. (Heating in the MegaByte chambers between construct additions and ligations denatures the enzymes to allow for proper conditions for MegaByte assembly.) The newly made MegaByte is pumped into the electroporation chamber where the MegaByte is transformed into a cell and ready for extraction.