Team:Glasgow/Project/Switch

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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.

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.

Gel One:

Figure 1: Gel 1, Lane Experiment. Cells grown in glucose or arabinose

1. pZJ7 on its own
2. Switch #2 on its own
3. Switch #2 + pZJ7 glucose
4. Switch #2 + pZJ7 arabinose
5. Switch #3 on its own
6. Switch #3 + pZJ7 glucose
7. Switch #3 + pZJ7 arabinose
8. Switch #4 on its own
9. Switch #4 + pZJ7 glucose
10. Switch #4 + pZJ7 arabinose
11. pBAD33 gvpAC (ignore)
12. 1kb+ marker

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.

Gel 2:

Figure 2: Gel 2 showing supercoiled DNA

2. Switch #2 + pZJ7 glucose
3. Switch #2 + pZJ7 arabinose
4. Switch #3 + pZJ7 glucose
5. Switch #3 + pZJ7 arabinose
6. Switch #4 + pZJ7 glucose
7. Switch #4 + pZJ7 arabinose
8. Switch #2 on its own
9. Switch #3 on its own
10. Switch #4 on its own
11. PZJ7 only

This gel shows uncut supercoiled vectors.
Switch vectors are around 2kb and integrase vectors around 3.4kb.

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 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 050914