Our Plans

First we want to note that our initial plans were very different from what we actually managed to do. Our plans were really amazing: we wanted to create system of biological machines which would allow to investigate protein interactions and simultaneously change sequence of one of them in order to achieve the strongest possible interaction. To make it work we planned to put the DNA sequence of tested protein on low-copy plasmid and transform it to bacterial strain in which it would be mutated (lets call the strain ‘slot machine’). Parts present on this plasmid would cause the protein to be attached to outer bacterial membrane, where the selection would occur. We planned that such selection system would allow us to search for antibodies with new specificities or screen protein libraries.

‘The slot machine’

Our goal is to change protein sequence in order to maximize its interaction with a given partner. Additionally we want to apply selection pressure on bacteria population so that only cells coding strongest interacting proteins survive. The solution of the first part of the problem is ‘the slot machine’ strain, which will randomize target protein sequence – nucleotides would be shuffled randomly in the same way it happens in popular hazard game. The simplest ‘slot machine’ strain is one of standard mutator E. coli strains without polymerase error correction activity or strain without DNA repair systems. It’s not optimal though - high mutation frequency in the whole bacterial genome would introduce some variance into our sequence, but would also cause problems with selection. Instead screening for protein interactions we would most likely obtain selection-resistant strain. So we wanted to narrow scope of mutations to a small well defined DNA fragment preferably carried on plasmid. Since at the beginning of our project we focused on antibodies the idea of using AID (Activation Induced Deaminase) protein came right away.

The AID protein is active in mammal lymphocytes, where it causes the somatic hypermutation – an increase of mutation level in antibody coding sequences. Moreover there was publication [PMID:12097915] which proved AID activity in E. coli cells. But that wasn’t yet what we wanted because AID mutated all highly-transcribed E. coli genes. We needed to find a way to target it to a specific DNA region. AID prefers single-stranded DNA that appears in highly-transcribed loci. So we needed to make our DNA sequence a highly transcribed one (preferably achieve the highest transcription level in the cell). Adding T7 promoter to our sequence seemed to be the perfect solution. Unfortunately many genes that can make cells selection-resistant are highly transcribed. So we went a step forward and created fusion between AID and T7 phage polymerase. T7 polymerase traverses the DNA fragment containing T7 promoter and carries AID, which introduces mutations.

AID is a small protein and its closest homologues form oligomers. The information about AID’s ability to form complexes is inconsistent, so we needed to consider such possibility and we created molecular device containing both free AID and AID-T7 fusion. We hope that AID-T7 fusion will recruit free AID to DNA sequence containing T7 promoter. To sum up we have created following molecular devices on pMPMT5omega plasmid under arabinose promoter:

1. AID

2. AID in translation fusion with T7 phage polymerase

3. AID in transcription fusion with T7 phage polymerase

4. AID in transcription fusion with AID-T7 translation fusion

To test various variants of AID we needed proper reporter system. We have used alpha-complementing beta-galactosidase fragment under control of T7 promoter. It was cloned to one-copy plasmid pZC30 (minireplicon of plasmid F). After obtaining cotransformants carrying one of AID devices and reporter plasmid and induction hoped to get some white colonies on X-gal plates indicating mutated clones.

Fig. 1. Our reporter system for checking site-specificity of AID-induced mutations
pBAD - arabinose promoter; pT7 - T7 promoter; TAXI=LB+Tetracycline+Ampicillin+IPTG+X-gal

Simultaneously we carried out the rifampicin test (plated liquid cultures of tested strains on plates containing 300 ug/ml antibiotic rifampicin) to check mutation level in whole genome of tested strains.

Fig. 2. Rifampicin test to check mutation level in bacteria expressing variants of AID.

In theory everything works nice but when we carried out the experiment reality was merciless. We obtained various numbers of white clones using different AID encoding devices but sequencing of beta-galactosidase gene from those clones revealed no mutations. It has to be a flaw in our reporter system. It seems that expression beta-galactosidase fragment encoded on pZC30 plasmid is somehow switched off – nobody knows why or how (we have sequenced large fragments of many white clones of pZC30 – no mutations, no clues). The test was carried out in two E. coli strains: Top10 and GM2163. The latter has damaged Dam and Dcm methyltransferases so DNA repair systems relying on their activity are unable to repair mismatches created by AID. The results obtained so far are presented in table below:

We are currently trying to create another reporter construct preferably based on different reporter gene (gfp or xyl) because it turned out that many strains have intact beta-galactosidase gene on chromosome. Until it’s ready our ‘slot machine’ will be one of E. coli strains with elevated total mutation level – most likely the mutD5 strain.

‘Hunter’ and ‘prey’

In former paragraphs we described our plans of introducing variation to protein coding sequence (lets call the protein ‘hunter’). Now it’s time to tell how we’ll be screening created population of ‘hunter’ proteins. At a first glance the most elegant solution is something similar to two hybrid system. But that requires putting both ‘hunter’ and ‘prey’ in the same cell of ‘slot machine’, which creates risk of changing ‘prey’ sequence - something we don’t want to happen. So the ‘prey’ protein must be supplied from outside and allow only best ‘hunters’ to survive.

This forces ‘hunter’ protein to be attached to cell surface. To achieve that all hunter constructs have fragment of OmpA (outer membrane protein) fused with ‘hunter’ protein. In this way ‘hunter’ is presented on E. coli cell surface, which allows it to interact with ‘prey’ added to liquid culture medium.

Fig. 3. Variants of the selection system using two parts of TEM-1 beta-lactamase (alpha and omega).

This is still not enough to ensure that only the best ‘hunters’ survive. Some selection pressure is needed to make only the best adapted survive. We have used TEM-1 beta-lactamase (protein responsible for resistance to beta-lactam antibiotics such as ampicillin) split into two complementing fragments: alpha and omega. Those fragments do not form active complexes spontaneously. Antibiotic resistance is achieved only when alpha and omega are in close proximity – i.e. when they are connected to two strongly interacting proteins. So ‘hunter’ protein connected with one beta-lactamase fragment will catch ‘prey’ protein connected to another and will allow survival of the cell in ampicillin containing medium. Cell line carrying the best hunter protein will have selection advantage over others.

In order to confirm that this system works we have chosen two small strongly interacting proteins A and Z. The A protein is the famous protein from Staphylococcus aureus which binds to constant fragments of IgG antibodies and has many uses in molecular biology. The Z protein is it’s artificially created close relative [PMID:15238637]. Apart from interacting with the A protein the Z protein has ability to form oligomers (this interaction is much weaker than with A though). We have created many variants of construct based on pACYC177 vector (low copy – 10 copies per cell) with IPTG-induced promoter. They contain OmpA fragment, A and Z proteins and beta-lactamase fragments in various combinations. The A protein consists of two nearly identical repeats (each one may interact with Z protein) so part of constructs contains truncated version of A protein (A delta). Lists of pACYC177 constructs:

1. OmpA_alpha

2. OmpA_omega

3. OmpA_A_alpha

4. OmpA_Z_alpha

5. OmpA_A_omega

6. OmpA_Z_omega

7. OmpA_omega_Adelta_alpha

8. OmpA_omega_Adelta

9. OmpA_Adelta_alpha

10. OmpA_Adelta_omega

We are working on construction of more pACYC177 derivatives.

The second group of constructs is used for overexpression and purification of ‘prey’. So far for this purpose we have used pET15b vector, which N-terminally His-tagged prey proteins. We have created:

1. His_Z_omega

2. His_Z_alpha

3. His_A_alpha

The first two constructs were successfully used for overexpression and purification of prey proteins. Use of His-tags and NiNTA beads was not needed because of Z protein oligomerization 90% of the protein was present in post-sonication debris. The work is still in progress but we have obtained some interesting results:

Our long-term plan is to replace A and Z proteins with antibody fragments and antigen but for now we need to check is it working. To sequence of the A protein we will introduce mutations which will make interaction with Z impossible. We will transform constructs carrying mutated A sequences to ‘slot machine’ strain – we want to check if and when reversion to wild type A will occur in mutant mixture. The simplest version of this experiment – mixing of two existing strains, isolating plasmid DNA and assessing strain proportions using restriction digests gave very promising results. After mixing equal amounts of OmpA_alpha and OmpA_A_alpha and using our selection system we have isolated only plasmid carrying OmpA_A_alpha (this experiment was also successful for mixture of OmpA_omega and OmpA_A_omega).

Further results and comments will be introduced in real-time. Stay in touch ;-)