Team:Glasgow/Project/Switch

The Switch

The switch is the central part of the project, and the key to making our new system function. We constructed the recombinase-activated switch using a terminator, a reverse BBa_J23100 promoter and spacers needed because Integrase works best with over 200 bases between att sites. We inserted the restriction sites, HinDIII and BamHI into the switch oligo. The size of the fragment changes when φC31 Integrase is able to recombine the att sites making it clear on a gel whether the DNA has been recombined or not.
What else did we have to do?

• A reverse RFP made using PCR to attach the prefix and suffix backwards reversing the BBa_E1010 ORF.
• Used reverse RBS BBa_I742130 and made new more efficient reverse RBS using Ribosome Binding Calculator. (Insert results of how G-RBS is better)
• Used B0034 upstream of E0040 GFP (recreating BBa_J85201) and cloned downstream of the recombinase switch (Part)
• Cloned Reverse RBS-RFP construct upstream of the recombinase switch (PART) + BBa_J8201 construct
  This construct was cloned into a low copy number vector with a PSC101 origin. The nature of a switch requires a low copy number within a cell to avoid ‘leakage’ of the signal. This leakage is caused by some RFPs being transcribed when GFP should be or vice-versa. Therefore having fewer plasmids in a cell reduces the leakage problem.

Once created, the RFP –switch – GFP construct needed to be placed into the vector best suited for it. We needed to put our construct into a low copy number plasmid, as the efficiency of the switch flipping would increase. We decided on a plasmid with an ORI of psc101 and carrying a kanamycin resistance gene, as the expression plasmid for the Integrase had chloramphenicol resistance. We didn’t have an empty plasmid available, so we had to modify PZJ53B. After removing what the plasmid was previously used to carry, an iGEM multiple cloning site was inserted. This MCS was of our own design, and consisted of the prefix and suffix, with a spacer in the middle containing a HinDIII site.

We next attempted to make the plasmid biobrick compatible. In the origin there was a Spe1 site, and we wanted to remove this. There were also two Pst1 sites on either side of the kanamycin resistance gene, but it would have been difficult to remove as it was flanked by long sequences of repeats. We designed a mutagenic oligo for the Spe1 site, attempts were made to remove the site by mutagenic PCR. Unfortunately, this did not work, possibly because of the changes this rendered to the origin. A previous iGEM team had removed this site, but it resulted in the plasmid no longer being a low copy number, and instead had a great variability in the number of copies each cell contained. The change we attempted was different from the previously attempted one and was more conservative, but in the end it didn’t work. We were left with the problem of having a plasmid that we couldn’t cut with Spe1 or Pst1, as it would cut outside of the MCS.

A possible solution lay in cutting with EcoR1 and Xba1 in the plasmid, and EcoR1 and Spe1 for our construct. As the construct was very close to the size of the plasmid it was in (Psb1c3), this had to be cut with another enzyme in order to separate the bands - Sac1-HF. The cells we transformed into were of strain DH5α.

The Integrase

φC31 Integrase att sites were used within the recombinase switch. Prefix and suffix were added to the integrase gene and it was cloned into PSB1C3. The Integrase gene has an EcoRI site within it so in order to remove it a double-stranded oligo was designed to change the guanine at the beginning of the EcoRI site into a cytosine. This change did not alter the resulting protein. The vector was digested with PstI and EcoRI, removing the first 40 bases from the Integrase gene. This digest was run on a gel before the vector and Integrase fragments were extracted and purified. The double-stranded oligo was ligated to these fragments, reconstituting the Integrase gene plus the prefix, making it a biobrick. The Integrase gene was cloned downstream of an arabinose-inducible promoter, pBAD33. φC31 Integrase recombination events can be reversed using the recombinase directionality factor, gp3. This gene was cloned into pSB1C3, but all attempts to clone it downstream of a BBa_J23100 + BBa_B0034 contruct failed, so the gene was not tested for functionality.

This φC31 Integrase biobrick was co-transformed into DH5α cells with the switch construct (PART). This allowed in vivo switching capabilities to be tested by looking at the fluorescence produced after the Integrase was induced. φC31 Integrase was induced using the sugar arabinose. The reason for this was because the pBAD33 promoter is well described and could be used as a proof of concept. In the future it is hoped that any inducible promoter can be used to switch on Integrase expression.

The fluorescence results showed no changed after Integrase expression in this strain. However it was later transformed into the E. coli strain DS941 As before, the experiment was done by putting two plasmids (pSC101 biobrick switchand a non-biobrick compatible version of Integrase, pZJ7) into E.coli, strain DS941.

The gel below shows what happens to the DNA when the cells were grown in glucose (no Integrase) or in arabinose (Integrase expressed). The experiment was carried out in triplicate, all worked equally well.

All are cut with BamHI.
pZJ7 gives the biggest band about 7kb
The unrecombined switch gives two bands about 2.4 and 2.7 kb
The recombined switch gives two bands about 2.5 and 2.6 kb.

The first fluorescence scan (040914-fluorescence below) shows the exact same  cells the DNA was extracted from in the same order as lane 2-11 on gel 2 above, and finally pBAD33 gvpAC as a negative control.
(red, green, red, green , red, green, red, red, red, almost black,  almost black). The two rows are just two identical 200 ul samples of each to show any pipetting errors or flecks of dust in the wells.

On the second scan (050914-fluorescence below), the top two rows are the same samples as on the first scan, but now one day old.

The next two rows show the same samples now grow another 10 generations  (overnight) in glucose. The ones that had not recombined, still fluoresce red, the ones that had recombined stay recombined and fluoresce green. So the system "remembers" that it has been exposed to arabinose.

The final two rows are the same as the two above, but they have been grown overnight with no glucose. Everything remains the same but the fluorescence is a bit brighter than the glucose samples, probably  because glucose reduces the switch plasmid copy number. These results prove our concept. If this switch is placed between two other genes, such as flagella and vesicle genes, it should in theory turn off flagella formation when the recombinase is induced by any substrate, turning on vesicle genes. This would stop the cells swimming and float them to the top of the media.

In Vitro Characterisation of the Switch

The switch was on a Cmr plasmid, and unless otherwise stated was the selective marker used throughout characterisation of the switch.

Part 1
6 eppendorfs containing 5ul of 4xIRB5 buffer, 2ul plasmid DNA, 11ul of ddH20 and 2ul of integrase were prepared. 3 containing GFP32 + Switch and 3 with GFP34 + Switch. The 32 and 34 denote different ribosome binding sites, with 34 being stronger. Into these 6 tubes went different concentrations of purified integrase – 4, 2 and 0uM. The 0uM consisting of the integrase buffer only. This reaction was incubated at 30DC for two hours.

Restriction digest of Integrase reaction
Chemical	Quantity
Integrase reaction	16um
100mM MgCl2	3ul
ddH20	10ul
HinDIII	0.75ul
PstI	0.75ul

A restriction digest was then performed on these six reactions using HinDIII and PstI. These enzymes were chosen because if the switch did indeed flip it would give different fragment sizes, and therefore a different banding pattern. Taking into account the requirements of non-HF HinDIII and the salt content of the integrase reaction, a specific digest to suit the needs of both enzymes was calculated.

25ul of the resulting reactions was then run on a gel to visualise (figure 8). This showed that roughly 10% of the switches had switched. The topmost of the altered bands is harder to see, owing to the larger size. However, the smaller fragments produce more a more visible difference.

1ul of these initial integrase reactions was transformed into TOP10 cells, in an effort to visualise the GFP. The GFP34 + 4uM integrase is shown below, after visualisation with A UV light. Some colonies can be seen to be lighter, though considering the low level of switching, this is not surprising.

Part 2
The procedure of the first set of procedures was repeated, changing the three concentrations of the integrase to 8, 4 and 0uM respectively. Now acquired HF HinDIII and IRB3 buffer for the integrase reactions, so the procedure for the reactions take place accordingly:

Edit