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Team:Glasgow/Project/Switch
From 2014.igem.org
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The Switch
The switch is the central part of the project, and the key to making our new system function. The idea is to place a promoter between two recombination sites: PhiC31 attB and attP. The two sites are in inverted (head-to-head) orientation so that expression of the integrase protein will flip the orientation of the DNA segment containing the promoter. Recombination between attB and attP produces two new sites (attL and attR) which are not recombined by the integrase, so the switching is unidirectional. We constructed the recombinase-activated switch using (BBa_K1463000) a reversed version of the BBa_J23100 promoter. Prior to recombination, this will direct transcription of genes to the left of the switch. After recombination, genes to the right will be transcribed. We incorporated a terminator to the right of the reverse promoter to prevent any read-through transcription of the genes we wish to stay OFF. We also incorporated spacers containing HindIII and BamHI sites, to make sure the att sites were far enough apart (>200bp) for efficient recombination and to allow us to monitor recombination by gel electrophoresis.
The following section will cover in vivo testing of the switch.
Click HERE to jump to the in vitro section of the report.
Click HERE to jump to the Integrase section of the report.
Click HERE to jump to the analysis of the switch Promoter.
What else did we have to do? The genes to the left of the switch have to be transcribed from right to left. We therefore had to come up with our own standard for reversed open reading frame biobricks and design an efficient reverse ribosome binding site (RBS) biobrick.
- Made a reverse RFP biobrick (BBa_K1463520) of BBa_E1010 by using PCR to attach the standard prefix just after the bar code of BBa_E1010 and the standard suffix just before the ATG start codon. Standard biobrick assembly will then give a reversed version of the BBa_E1010 ORF.
- Tested the reverse RBS BBa_I742130 and our own more efficient reverse RBS (BBa_K1463560) using Ribosome Binding Calculator. (data not shown)
- Used B0034 upstream of E0040 GFP (recreating BBa_J85201) and cloned downstream of BBa_K1463050
- Cloned Reverse RBS-RFP construct upstream of BBa_K1463050 + 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 attP and attB sites (BBa_K1463040 and BBa_K1463041) were used within the recombinase switch, therefore we needed the PhiC31 integrase to control switching. To make a PhiC31 integrase biobrick, prefix and suffix sequences were added to the integrase gene by PCR 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 in the plasmid pBAD33 (Guzman et al 1995 PMID: 7608087).
The directionality of φC31 integrase recombination can be reversed using the recombinase directionality factor, gp3 (Khaleel et al 2011 PMID: 21564337). This protein allows the integrase to recombine attL and attR, thus potentially reversing the directionality of our recombination swithch. φC31 gp3 gene was cloned into pSB1C3, but all attempts to clone it downstream of a BBa_J23100 promoter + BBa_B0034 RBS failed, so the gene was not tested for functionality.
This φC31 integrase biobrick on pBAD33 was co-transformed into DS941 cells with the switch construct (BBa_K1463000) carried on a low copy number pSC101 vector. This allowed in vivo switching capabilities to be tested by restriction digestion of the switch DNA and by looking at the fluorescence produced. φC31 integrase was induced using the sugar arabinose. The arabinose inducible promoter on pBAD33 promoter is well described and tightly regulated and was therefore ideal as a proof of concept. In the future it is hoped that any inducible promoter could be used to switch on integrase expression only in the desired conditions.
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. There was no change in restriction pattern of the switch plasmid when cells were grown in glucose, while all of the switch plasmid changed to the expected restriction pattern when the cells were grown on arabinose.
Gel One:
Figure 1: Gel 1, Lane Experiment. Cells grown in glucose or arabinose
- pBAD-int on its own
- Switch #2 on its own
- Switch #2 + pBAD-int glucose
- Switch #2 + pBAD-int arabinose
- Switch #3 on its own
- Switch #3 + pBAD-int glucose
- Switch #3 + pBAD-int arabinose
- Switch #4 on its own
- Switch #4 + pBAD-int glucose
- Switch #4 + pBAD-int arabinose
- pBAD33 gvpAC (ignore)
- 1kb+ marker
All are cut with BamHI.
pBAD-int 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.
Gel 2:
Figure 2: Gel 2 showing supercoiled DNA
- Switch #2 + pBAD-int glucose
- Switch #2 + pBAD-int arabinose
- Switch #3 + pBAD-int glucose
- Switch #3 + pBAD-int arabinose
- Switch #4 + pBAD-int glucose
- Switch #4 + pBAD-int arabinose
- Switch #2 on its own
- Switch #3 on its own
- Switch #4 on its own
- pBAD-int only
This gel shows uncut supercoiled plasmid DNA from the same assay. Addition of arabinose leads to DNA inversion which does not change the size of the switch plasmid, so no change is seen. However a reduction in plasmid yield is noticeable in the presence of glucose.
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.
Figure 3:Fluorescence Scan of 200 ul samples of overnight cultures in a 96 well plate using a Typhoon FLA9600 scanner. Overlay of red and green fluorescent images using 575 nm laser and BPFR filter and 450nm laser and LPB filter respectively. (Scan 040914)
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.
Figure 4: Fluorescence Scan produced as in figure 4. (Scan 050914)
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 (see gel). These results prove our concept. If this switch is placed between two other genes, e.g. with flagellar or other motility genes on the left and gas vesicle genes on the right, 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.
Efficiency of Switch Promoter
In order to determine how efficient the switch promoter is, in comparison to the J23100 promoter without the att sites, a plasmid was generated with J23100 promoter upstream of the GFP with the BB0034 RBS. The fluorescence of transformants containing this plasmid was compared with the fluorescence of transformants containing K1463000 (RFP/switch/GFP) exposed to integrase which fluoresce green (figure 5). Measuring the fluorescence levels revealed that the switch promoter is less efficient compared to the J23100 promoter. This suggests that the att sites and intervening sequences between the promoter and the RBS interferes with the strength of the promoter.
Figure 5: Relative Fluorescence of
Figure 6: Relative Fluorescence of
In Vitro Characterisation of the Switch
Figure 7:
The switch was on a Cmr plasmid, and unless otherwise stated was the selective marker used throughout characterisation of the switch.
Figure 8:
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.
| Integrase reaction | |
|---|---|
| Chemical | Quantity |
| 4xIRB5 buffer | 5ul |
| Plasmid DNA | 2ul |
| Integrase | 2ul |
| 0.1mg/ml BSA | 11ul |
| IRB5 Buffer 1x 1ml | |
|---|---|
| Chemical | Quantity |
| 50mM Tris 7.5 | 200ul |
| 5mM Spermidine | 40ul |
| 0.1mg/ml BSA | 40ul |
| H20 | 11ul |
Figure 9:
| 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.
Figure 11:
Figure 10:
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:
| Restriction Digest of Integrase Reaction | |
|---|---|
| Chemical | Quantity |
| Integrase reaction | 16ul |
| Cutsmart | 3ul |
| ddH20 | 10ul |
| HF HinDIII | 0.75ul |
| HF PstI | 0.75ul |
| Integrase Reaction | |
|---|---|
| Chemical | Quantity |
| IRB3 buffer | 5ul |
| Plasmid DNA | 2ul |
| Integrase | 2ul |
| H20 | 11ul |
Figure 12:
Figure 13:
1. Switch & GFP34 – 8uM int. 2. Switch & GFP34 – 4uM int. 3. Switch & GFP34 – 0uM int.
4. Switch & GFP32 – 8uM int. 5. Switch & GFP32 – 4uM int. 6. Switch & GFP34 – 0uM int.
7. Switch & GFP34 – 8uM int. 8. Switch & GFP34 – 4uM int. 9. Switch & GFP34 – 0uM int.
10. Switch & GFP32 – 8uM int. 11. Switch & GFP32 – 4uM int. 12. Switch & GFP34 – 0uM int.
The procedure was repeated generating two sets of reactions, seen above. The results this time were much clearer, showing that the switch exposed to the integrase does correctly switch, generating the fragment sizes expected.
From this, TOP10 cells were again transformed with 1ul of the integrase reaction. 6 plates for each reaction - GFP32 with 8, 4 and 0uM integrase; GFP34 with 8, 4 and 0uM integrase – were grown overnight and visualised. The results of this are shown in figure 12.
It can be seen that some of the colonies are indeed fluorescing, but none of the 0uM are, indicating that the switch flipped correctly and only after the exposure to the integrase. Those transformants containing a plasmid with a flipped switch will show up fluorescent, and those that don’t, will not. This should be visible by isolating the plasmid from the colonies and performing a restriction digest on the said plasmids. However, the transformation proved highly successful, and the colonies grew too close together, and more resembled a lawn. Picking with accuracy therefore proved difficult. Transformations were repeated using 50ul of cells during the addition of the DNA, instead of 100ul. As well as re-transforming the first set of integrase reactions, the second set (the repeat present on the gel) was also transformed. The results of this can be seen in figure 13.
Figure 14: Plate Visualisation. The colonies present on each plate are:
1. GFP32 + 0uM int. 2. GFP32+ 4uM int. 3. GFP32 + 8uM int.
4. GFP34 + 0uM int. 5. GFP34 + 4uM int. 6. GFP34 + 8uM int.
Figure 15: Plate Visualisation. The colonies present on each plate are:
1. GFP34 + 8uM int. (1) 2. GFP34 + 8uM int. (2) 3. GFP32 + 8uM int. (1) 4. GFP32 + 8uM int. (2)>br>
5. GFP34 + 4uM int. (1) 6. GFP34 + 4uM int. (2) 7. GFP32 + 4uM int. (1) 8. GFP32 + 4uM int. (2)
9. GFP34 + 0uM int. (1) 10. GFP34 + 0uM int.(2) 11. GFP32 + 0uM int. (1) 12. GFP32 + 0uM int. (2)
Figure 16:
Selecting colonies
From this, 20 colonies each were selected from both the GFP34 +8uM int. and GFP34 + 4uM plates both the at random, as distinguishing fluorescing from non-fluorescing colonies in natural light proved difficult. These were short streaked out onto fresh agar plates.
The colonies marked black 1-4 were expected to have the plasmid in the switched form, and the colonies marked white 1-4 were expected to have the plasmid in the un-switched form. The two colonies marked grey were those that seemed to be fluorescing but at a lower level than the others. It was hypothesised that this was either due to the cells having taken up both a switched and an un-switched plasmid; or have a switched plasmid but at a lower copy number than the other colonies. The required restriction digest for the plasmid taken from the colonies does not require special consideration to be made of the DNA content and the salt content of the integrase buffer and proceeds as normal.
| Restriction Digest | |
|---|---|
| Chemical | Quantity |
| 10x Cutsmart buffer | 2ul |
| Plasmid DNA | 4ul |
| ddH20 | 14ul |
| HF HinDIII | 0.75ul |
| HF PstI | 0.75ul |
It can be seen in the gel below (figure 17) that all of the colonies marked B1-4 are in the switched form as expected, and all those marked W1-4 are in the un-switched form. This is as expected, and shows that the switch does indeed correctly switch in the presence of integrase, and that the now correctly orientated promoter does indeed drive expression of GFP. It can also be seen in the colonies marked G1 & G2 that both the hypotheses with how the greyer colonies could result appear to be correct. G1 shows the presence of two bands, indicating the colony had both a switched and un-switched plasmid. G2 on the other hand shows one band indicating the presence of a flipped switch. However the bands in this lane are generally fainter that the others, which could have resulted from an error during preparation of the DNA. However, as the DNA came from a less-brightly fluorescing colony, it could be the result of the plasmid carrying the switch being present at a lower copy number.
Figure 17:
From this, it can be seen that exposing the switch to φC31 integrase does result in a successful switching which will drive expression of the downstream GFP. It can also be seen that in a high copy number plasmid such as PSB1C3 this switching can be less clear-cut than desired, as it is possible for the cells to carry both switched and un-switched versions of the plasmid, suggesting the need for a low copy number plasmid.
