Team:Edinburgh/Project/Protocol

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BRIDGE: The concept

BRIDGE stands for BioBrick Recombination In Direct Genomic Editing. It is an alternative method for inserting BioBricks into the genome by using homologous recombination instead of restriction digestion, with the added bonus of not leaving a marker behind in the product.

The first step of BRIDGE requires the deletion of existing DNA (probably a non-coding piece or a non-essential gene) to introduce a construct of two genes; one an antibiotic resistance gene, the other sacB, which prevents the host from growing on sucrose. After the first step we can select for cells which have taken up the construct by growing them on the relevant antibiotic.

The second step involves swapping the construct for another piece of DNA (e.g. a BioBrick construct). After this we can select for those with the new gene by growing the cells on sucrose. Neither selection marker is left in the genome, but the original DNA is replaced with the desired insert (or simply deleted).

BRIDGE: The advantages

BRIDGE has a significant advantage over the current method of BioBrick insertion. For one, it is vector independent - whole PCR constructs can be inserted directly into the genome in two steps in under a week, compared to the lengthy process of vector digestion and ligation required with normal BioBricks.

The other major advantage is that it will not leave a lasting marker in the genome. With most BioBricks we have to leave a marker (antibiotic resistance, GFP, etc) in our constructs so that we can guarantee their presence. This becomes an issue, a) when you want to use the organism in an industrial or environmental capacity, and b) when you want to insert multiple constructs into the same host organism (there are only a limited number of markers out there). With this system, the markers are removed every time a new gene is inserted, so they can be used again and again indefinitely. You could essentially replace the entire genome with new genes, should you be so inclined!

BRIDGE: The protocol

The idea here is that I will write up the method that gave us the best results in the shortest time. The protocol that is up here by the wiki freeze may not be the optimum, but will be based on our success so far. To see the progression of this protocol, visit the "BRIDGE" page in the lab notes drop down menu.

Introduction

The bulk of this protocol has been developed from the Gene Bridges "Quick and Easy BAC Modification Kit by Red/ET Recombination" protocol, version 2.4 from 2005. We have had to edit in places as we are not using vectors and have two strains of E.coli. For clarification on why certain steps are taken you should refer to the full protocol. If you are using an alternative plasmid then you should refer to a protocol relevant to this plasmid for sections 1 and 2 (i.e. growth conditions).
I will give you a brief overview of how to make your constructs by traditional biobricking methods and will then go into detail about how to transform and select for you recombinants.

The two E.coli strains we used were JM109 and DH5alpha. When transformed with the Red/ET these are tetracycline resistant, but you be aware that the two strains require different concentrations of tetracycline in liquid cultures as JM109 has greater antibiotic resistance than DH5alpha. These concentrations are given in the protocol but remember that if you alter the volume of you liquid cultures you should also alter the volume of tet15 added.
You should also bear in mind that the Red/ET plasmid will not replicate at 37C so will be lost from cells grown at 37C for more than an hour. For this reason we grew our transformants at both 30C and 37C to retain the plasmid and to gain decent growth for characterisation and determination of results.

For this experiment you will need negative controls for your transformations to confirm that growth is definitely due to the presence of the chloramphenicol resistance gene. We grew 1 liquid culture for each strain, prepared 2 transformations for each (one with and one without DNA), grew each transformant at both 30C and 37C and then plated each of these onto cml40 at both a high and a low concentration of cells. By the end of the first step you should have 16 plates: JM109/DH5alpha +/-DNA at 30/37C and concentrated/not concentrated.

Sections:
1- Creating homology between target and constructs
2- Preparing cells with Red/ET
3- Preparing cells for electroporation
4- Electroporation and recovery steps
5- Selecting for recombinants
6- Stating again for the second step


Materials: (these will be given again for each section)
- Ingredients and equipment required for PCR and restriction enzyme disgestion
- A good stock of sterile LB
- A large supply of agar plates with cml40
- Agar plates with tet15
- Tetracycline (15mg/ml)
- Chloramphenicol (40mg/ml)
- L-arabinose
- Access to electroporation technology
- Sterile electroporation cuvettes
- Sterile water
- Eppendorfs


Section 1 - Creating homology between target and constructs

In order to perform bridge, the cat/sacB construct (or whichever combination of markers you choose) and the gene you wish to insert must have homology with a region of the E.coli genome. This could be a non-essential gene, for example we used tnaA which codes for indole production, or it could be a non-coding, non-functional region of the genome.
Once you have chosen a section of DNA for deletion, you need to identify and obtain the flanking (upstream and downstream) sequences of that region. This is best done by designing and synthesising primers with the correct biobrick restriction sites for the two sequences and amplifying them directly out of E.coli by PCR. Alternatively you could have the full sequences synthesised, but this is less cost-effective.
The flanking sequences need to be biobricks, i.e. they should have EcoRI and XbaI sites at their upstream end and SpeI and PstI at their downstream end.

Once you have your flanking sequences you need to digest the marker construct (which should already be in biobrick format) with XbaI and your upstream sequence with SpeI before ligating them together. This should then be amplified using the upstream forward primer and the marker construct reverse primer.
You now need to redigest the new construct with SpeI and digest your downstream sequence with XbaI and ligate them. Using the forward primer of the upstream sequence and the reverse primer of the downstream sequence you can now amplify the final contrust from this ligation.
Repeat this process with the gene you wish to insert at the final step and clean both PCR products. These can no be used in the protocol.


Section 2 - Preparing cells with Red/ET

The initial protocol suggests an electroporation procedure for inserting the plasmid. We prefer to use a simple cold shock-heat shock method, which seems to work just as efficiently. To see the exact protocol used go to the lab notes drop down menu, click protocols then click "Preparing and using compotent E.coli cells".

Spread the transformants to tetracycline (15mg/ml) (or relevant antibiotic if not using Red/ET) plates and then subculture to a second plate as a stock of cells.

Section 3 - Preparing the cells for electroporation

1 - Prepare 5ml LB in vials with tetracycline at 15mg/ml (tet15) (NB: if using a less resistant strain such as DH5alpha, use a lower concentration of tetracycline)
2 - Pick cells from your stock plate and innoculate the vials
3 - Incubate cultures overnight at 30C with shaking (200rpm)
4 - Prepare another set of vials with 5ml LB and tet15
5 - Use 0.5ml of the overnight cultures to innoculate the new vials
6 - Grow the new liquid cultures at 30C for 2 hours with shaking (200rpm)

Control Experiments

This is a long experiment with much room for error and little for quantification. These additional experiments were run when we needed to confirm or rule out problems with the protocol. We recommend you use them in the same way to solve similar problems.

Contamination:
To be used for narrowing down stage of entry of contaminants.

For the first two runs of BRIDGE we attempted, the cultures became contaminated. One type of the contaminants was RFP containing E.coli, another was micrococcus (which probably came from the experimenter) and the third was an unidentified organism which grew into white, runny colonies and is apparently chlromphenicol resistant and prefers 30C to 37C. (See lab notes for details).
To determine where these were coming from we decided to take samples of the cells after every step of the protocol which could involve human error or required contact with another solution or object and plate them to chloramphenicol 40 plates. This allowed us to narrow down the source. If you suspect contamination on your final plates you might want to follow these steps in addition to the protocol above.

We took samples at 5 stages prior to the final spreading of the transformants:
1- After growing the strains overnight at 30C
2- After growing the cells for 2 hours at 30C in new LB
3- After growing the cells at 37C for one hour with arabinose
4- After cleaning the cells with sterile water
5- After growing for a couple of hours after electroporation

If the contamination appears after:
Sample 1- either the original strains are contaminated or they are getting by human error
Sample 2- they are probably getting in by human error
Sample 3- the arabinose is contaminated
Sample 4- either the sterile water or the eppendorfs are contaminated (or possibly too much exposure to non-sterile air)
Sample 5- contaminants are coming from the electroporation lab/area
The final spreading of the transformants - human error in spreading

Negative Controls:
To be used to pinpoint source of contamination.

The previous experiment narrowed down the contamination to the washing step. This meant it could either be the sterile water, the unsterile air or the eppendorfs.
The next time we performed the BRIDGE protocol we took 4 samples at the washing step:
- DH5alpha after being washed under the clean hood
- JM109 after being washed under the clean hood
- Sterile water from previous run of experiment kept in an eppendorf for ten minutes
- Fresh sterile LB kept in an eppendorf for ten minutes

Each were plated to cml40 plates.
If contamination occurs on:
- Bacterial samples only - samples were already contaminated
- Sterile water but not LB - sterile water is contaminated
- Both sterile water and LB - eppendorfs are contaminated
- None - using the clean hood as prevented contamination

We got no growth from these controls and samples. We concluded that the exposure to non-sterile air had lead to the contamination in the previous experiments.

Lethality of Electroporation:
To be used if cells are not growing in order to rule out death by electroporation.

At some point we became concerned that the electroporation was damaging the cells and preventing them from growing after transformation. To determine if this was the case we set up extra liquid cultures after the recovery step.
As well as the normal 8 cultures in chloramphenicol (see above) we set up each of the positive transformants (those electrporated with the cat/sacB construct) in tetracycline at the concentrations used in the initial overnight cultures (tet15/5microlitres for JM109 and tet3.75/1.75microlitres for DH5alpha).

Whereas none of the chloramphenicol-containing cultures grew, 3 of the tetracycline-containing cultures did (see lab notes for details).
This confirmed that, in the majority of cases, elecrtroporation was not killing the cells. We then had to conclude that either the Red/ET plasmid is incorrect (i.e. we were using the wrong one) or that the recombinase genes were not being properly induced.

Titre of Resistance
To be used for weak antibiotic resistance genes to confirm if a protocol works or to characterise said gene.

The cat gene we used for chloramphenicol resistance in this procedure is not a very strongly induced gene. When the cells still wouldn't grow in lowered concentrations of chloramphenicol (see lab notes) we decided to confirm if they could grow in the presence of any chloramphenicol. For this we designed and carried out a simple experiment which can be used to titre the antibiotic resistance of a gene.

Each transformant (JM109+cat/sacB and DH5alpha+) was grown in 5ml liquid LB cultures with increasing concentrations of chloramphenicol - 0(0microlitres) to 24(3microlitres) - along with non-transformant controls of both strains.

This experiment can be interperated both qualitatively and quantitatively:
- Qualitatively - determine the presence of growth by eye (if characterisation is unimportant)
- Quantitatively - measure the optical density of each culture relative to that of LB

From this we concluded that cat is definitely not present in the transformants.

BioBricks

BBa_K322210: chloramphenicol resistance gene (chloramphenicol acetyltransferase)

BBa_K322921: Bacillus subtilis levansucrase, lethal to E. coli in the presence of sucrose.

BBa_K322922: composite construct.

BBa_K322705: Upstream region of E. coli tryptophanase locus, used for targetting genes to this locus using BRIDGE.

BBa_K322706: Downstream region of E. coli tryptophanase locus, used for targetting genes to this locus using BRIDGE.

Characterisation

It works!

References

Sun, W., Wang, S. & Curtiss, R. (2008). Highly Efficient Method for Introducing Successive Multiple Scarless Gene Deletions and Markerless Gene Insertions into the Yersinia pestis Chromosome Appl Environ Microbiol. 2008 July; 74(13): 4241–4245.