The objective of this experiment was to synthesize a new plasmid concatenated from two different BioBricks. BioBricks are pre-assembled plasmids, which are composed of genes of interest flanked by certain conserved DNA sequences. We used restriction enzymes, which cut DNA at the specific sites mentioned above, to cut each plasmid in such a way that both can be ligated together to produce a single plasmid containing all of the genes of interest.
When certain restriction enzymes cut DNA, they leave an overhang on one of the two strands. These overhangs are known as "sticky ends" because they can be paired with complementary overhangs and ligated together. XbaI and SpeI, two restriction enzymes, cut in the following manner:
XbaI: T C T A G A -- becomes --> T C T A G A A G A T C T A G A T C T
SpeI: A C T A G T -- becomes --> A C T A G T T G A T C A T G A T C A
These two enzymes both recognize unique 6-base sites, and also happen to share the same overhang (CTAG/GATC). However, when an XbaI end and a SpeI end are ligated together, the resulting construct has neither an XbaI nor an SpeI site:
C T A G A T A C T A G A + -----> T G A T C T A T G A T C
This allows plasmids made of many BioBricks to be assembled serially using the same protocol.
There are two methods of serial assembly: prefix and suffix insertion. Prefix insertion places the new genes in front of the old genes in the target plasmid; suffix insertion places them after.
In this diagram, E represents the EcoRI site, X the XbaI site, S the SpeI site, and P the PstI site. The blue, green and gray blocks represent genes; red blocks represent termination sequences; M the mixed restriction sites resulting from previous XbaI/SpeI ligations.
Prefix insertion occurs when an fragment of another plasmid is inserted into the vector plasmid between the EcoRI and XbaI sites. To do so, the vector plasmid is cut with EcoRI and XbaI, while the insert plasmid is cut with EcoRI and SpeI. The insert fragment is then ligated into the vector plasmid as shown:
|---E E---X---Insert-Gene---S X---Vector Gene---S---P---|
Note that the SpeI site and the XbaI site ligate together.
Suffix insertion occus when the fragment is inserted between the SpeI and PstI sites. This is achieved by cutting the vector plasmid with SpeI and PstI, and the insert plasmid with XbaI and PstI. The insert fragment is then ligated into the vector plasmid as shown:
|---E---X---Vector-Gene---S X---Insert-Gene---S---P P---|
Suffix insertion is generally preferable to prefix insertion because EcoRI is sometimes too efficient at cutting DNA, and cuts even where there is no restriction site: if both BioBricks are large (around 500 bp and up), use suffix insertion. You will need to use prefix insertion if the second BioBrick is small (under 200 bp), because differences of this size are hard to resolve on a gel and one risks the insert running off the edge.
(If both pieces are under 100 kb, one might be able to digest for suffix insertion without gel extraction, instead knocking out the Amp resistance cassette from the insert with XmnI and PCR purifying before ligation. Using NEBCutter, make sure that XmnI doesn't cut in the middle of the BioBrick, as it cuts with blunt ends.)
Protocol for BioBrick Insertion
1. Set aside two Eppendorf tubes. Label one "Vector" and one "Insert."
2. Get aliquots of 10X BSA, NEBuffer 2 and NEBuffer 3, XbaI, SpeI and PstI from the 4° fridge in room 5096 (on the right as you enter). Keep the enzymes on ice and thaw the BSA and buffers at room temperature or by hand.
3. Make master mix (vortex BSA and Buffers before pipetting), for each mix:
- 1 uL 10x BSA
- 1 uL 10x Buffer NEB(3 for vector, 2 for insert)
- 0.2 uL enzyme 1 (SpeI for vector, XbaI for insert)
- 0.2 uL PstI
- 5.6 uL distilled water to make total volume 10 uL with the DNA
4. It is ok to vortex the master mix gently.
5. To 8 uL master mix, add 2 uL DNA (700 ng plasmid (vector or insert)). Vortex gently and briefly spin down.
6. Incubate tubes at 37 °C overnight.
7. CIP vectors. Add a drop of CIP to each reaction mixture (to get a drop, put a pipet tip in the tube of enzyme and then quickly withdraw it, leaving a small drop on the tip -- this is done because we don't have a pipet that goes below 0.5λ).
8. Gel extract (run on agarose gel and use Qiagen Gel Extraction kit), and then proceed to ligation.
In Step 3, cut the vector with EcoRI and XbaI and the insert with EcoRI and SpeI. Vector should be digested in NEB buffer 2, while insert should be digested in EcoRI buffer.
Protocol for Analytical Digestion
Follow above protocol, adding 2 µl of DNA from miniprep (assuming it was brought up in 20 µl). To assess size of BioBrick, use XbaI and SpeI. Use NEB2 buffer in master mix. Run results on agarose gel.