Edinburgh/Yoghurt/Wet Lab

From 2007.igem.org

BioBrick Creation

Overview of the process we used to generate our BioBricks:

Figure 1

1. Designed primers, which included a ribosome binding site and the BioBrick restriction sites, generally SpeI and either EcoRI or SacI (for use with Edinbrick vectors, which have a SacI site overlapping the XbaI site) to amplify our desired gene out of its host organism.

2. Purified the PCR product and loaded onto an agrose gel to check for the presence of PCR product of the right size.

3. If a band was present at the right size, we digested the PCR product and vector (usually Edinbrick 1, which allows blue white selection) with the appropriate restriction enzymes.

4. Digested PCR product and vector were ligated overnight with T4 DNA ligase.

5. Ligated vector and gene were then transformed into E. coli and plated onto Blue/ White selection Amplicillin plates.

6. Transformants that contained a product ligated into the vector would grow as white colonies and are easy to select.

7. Minipreps were prepared of the white colonies.

8. Purified vector DNA was digested with restriction enzymes EcoRI and PstI to determine if the vector insert was of the correct size.

9. Vectors containing inserts of the correct size were sent for sequencing to confirm if they did indeed contain the correct gene.

The complete methods we used to generate our BioBricks may be found on the French Lab OpenWetWare site.

Zeaxanthin Synthesis Pathway

BioBricks created so far

crtE (BBa_I742152)
crtIB (with PstI restriction sites)(BBa_I742161)
crtZ (BBa_I742158)
crtY (BBa_I742155)

Primers

Gene	Forward Primer	Reverse Primer
CrtE	crtE f1: gca gagctc gcgt tgcc gtaa atgt atc	crtE r2: cgt actagt a tta tta act gac ggc agc gag
CrtBI	crtI f1: tga gagctc atcg ttaa agag cgac	crtB r2: cgt actagt a tta tta gag cgg gcg ctg cca g
CrtY	crtY f1: cag gagctc ttaa gtgg gagc ggct atg	crtY r2: cgt actagt a tta tta acg atg agt cgt cat aat g
CrtZ	crtZ f1: aga gagctc tacc ggag aaat tatg	crtZ r2: cgt actagt a tta tta ctt ccc gga tgc ggg

More information on the primers used to amplify the zeaxanthin pathway genes may be found on the French Lab OpenWetWare site.

The crtIB BioBrick was subjected to site directed mutagenesis to remove the two PstI sites. A version with the PstI sites successfully removed was generated. However, the final step, required to add a compliant BioBrick suffix to this mutated gene, was not successful and thus the mutated version has not yet been submitted to the Registry. We hope that this will be completed next year by an Honours project student in the laboratory of Dr. Chris French.

Lycopene BioBrick

Success at last!

Figure 2. E. coli colonies that have started to synthesise lycopene

We have managed to successfully clone crtE and crtIB genes and ligate them together in a BioBrick, which produced a pink pigment, possibly lycopene. To confirm the identity of this pigment, it will be necessary to compare it with authentic lycopene using analytical techniques such as spectral analysis, thin layer chromatography, HPLC, and possibly mass spectrometry.

Unfortunately, since this construct used the version of crtIB with the PstI sites still present, it was not considered appropriate to add crtY and crtZ until the mutated verrsion of crtIB, lacking the PstI sites, was available. This was unfortunately not achieved within the time scale of the project. However, this project has been offered as an Honours research project in the laboratory of Dr. Chris French, so will be continued next year.

Figure 2 shows some E. coli colonies, which have produced a small amount of red pigment.

Lycopene BioBrick in our sandox in the Registry BBa_I742120 and BBa_I742136

Beta-carotene BioBrick

Beta-carotene BioBrick in our sandbox in the Registry BBa_I742121 and BBa_I742138

Zeaxanthin BioBrick

Zeaxanthin BioBrick in our sandbox in the Registry BBa_I742139 and BBa_I742122

Vanillin Biosynthesis Pathway

BioBricks created so far

Genes sent to GENEART for synthesis

Primers

Gene	Forward Primer	Reverse Primer
Sam5	sam5 f1: at gaattc gcggccgc t tctag atg acc atc acg tca cct g sam5 f2: tc gaattc gcggccgc t tctaga g acggagaagcagcgaaatg	sam5 r1: ct actagt a tta tta ggt gcc ggg gtt gat cag
Sam8	sam8 f1: at gaattc gcggccgc t tctag atg acg cag gtc gtg gaa cg sam8 f2: tc gaattc gcggccgc t tctaga g gaggttgggacaagatgac	sam8 r1: ct actagt a tta tta tcc gaa atc ctt ccc gtc
ech	ech f1: aat gaattc gcggccgc t tctag atg agc aaa tac gaa ggt c ech f2: atc gagctc acacc cagaa caaga gc	ech r1: ct actagt a tta tta gcg ttt ata ggc ttg cag c ech r2: tt actagt atcgg gaaca cgttc aagc
fcs	fcs f1: aat gaattc gcggccgc t tctag atg cgc tcc ctg gaa ccc fcs f2: gtg gagctc actga agaac agggc gtg	fcs r1: ct actagt a tta tta cgg ttt ggg ccc ggc ac fcs r2: aa actagt atgcc gtgac agcaa atagg

More information on the primers used to amplify the vanillin pathway genes may be found on the French Lab OpenWetWare site

Cloning of ech and fcs genes from Pseudomonas fluorescens NCIMB9046 (JCM5963)

We decided to clone ech and fcs by directly amplifying the genes from the P. fluorescens genome. In order to do this we ordered two primer sets (one for each gene), which also encoded the relevant BioBrick restriction sites. In addition, the ech reverse primer was modified to point mutate out the PstI restriction site found in the genes 3' end.

After trying many different annealing temperatures, we found that the two genes were not being amplified up by PCR. This could be due to the high GC nature of the P. fluorescens genome. Therefore we decided to modify the PCR reaction with glycerol or DSMO, which are both reported to improve PCR amplification of GC rich organisms. In addition we also designed a second primer set, by searching for sites further away from the gene with a lower GC content, which hopefully would improve primer binding. The second primer sets also contained a ribosome binding site.

The use of the second primer set and glycerol resulted in a PCR product for the fcs gene of ~2kb. This fcs PCR product was then purified, digested with SacI/ SpeI restriction enzymes and ligated into the Edinbrick1 vector. So far we have not yet managed to produce any fcs clones with a viable fcs insert.

Unfortunately there was no viable PCR product for the ech gene. Therefore we decided to send a modified ech gene to GeneArt for synthesis. We modified the ech gene by altering the coding reading frame to have the optimum codons for E. coli; this was both to decrease the GC content of the gene and to ensure optimal expression of ech. Later, we also ordered fcs on the same basis. Unfortunately, due to the late stage at which it became apparent that PCR would not yield results, neither ech nor fcs is currently available. However, this project will be continued by an Honours project student, so we hope that the ech and fcs BioBricks can be submitted to the Registry at that time.

Cloning of Sam5 and Sam8 genes from Saccharothrix espanaensis

Just as with the ech and fcs genes, we designed primers to amplify up Sam5 and Sam8 from the S. espanaensis genome. Two primer sets were used for each gene. In both cases, f1+r1 should amplify the coding sequence alone, whereas f2+r1 should include the native ribosome binding site. EcoRI sites were used in the forward primers rather than SacI, since both sam5 and sam8 have internal SacI sites.

As with the ech and fcs genes, we also found that a simple PCR resulted in no PCR product, due to S. espeanensis also having a high GC genome (recognising a pattern here).

However glycerol and a higher melting temperature came to our rescue, and by incorporating these two factors into our PCR, we got products for both Sam5 and Sam8.

The PCR amplifications of the two genes were digested with EcoRI and SpeI restriction enzymes and ligated into the Edinbrick1 vector. Colonies containing either the Sam5 or Sam8 gene were easily identified and minipreps were made of their vector DNA. Due to shortage of time, two different versions of each gene were made: one being the coding sequence alone, the other being the coding sequence with the native ribosome binding site. We should also note that the Sam5 coding sequence contains a PstI site. Time precluded removal of this site by mutagenesis, but we hope that, if initial test experiments are successful, this mutation will be undertaken at a future time.

The purified vector DNA was then sequenced to confirm that the genes present were indeed Sam5 and Sam8. Fortunately for us the sequencing results confirmed we had isolated the right genes.

We then made a Plac-Sam8 construct, to induce Sam8 expression upon the addition of IPTG (BBa_I742144). A sam8-sam5 construct was also prepared (BBa_I742143).

The next stage is to test these constructs, to determine whether native proteins are synthesised and the correct enzymatic reactions are carried out. We plan to do this by inducing tyrosine ammonia lyase (sam8) and p-coumoryl-4-hydroxylase (sam5) in broth cultures, adding relevant substrates (tyrosine, p-coumaric acid) and incubating. Products will initially be detected by thin layer chromatography. We have demonstrated using authentic standards that the relevant compounds can be separated using silica plates with integral UV indicator with a mobile phase of 50:50:1 hexane-ethyl acetate-acetic acid. The Rf values are: coumaric acid 0.60 (black spot); caffeic acid 0.31 (purple spot), ferulic acid 0.57 (purple spot), and vanillin 0.74.

Gas chromatography coupled with mass spectrometry (GC-MS) will then be used to test for the production of p-coumaric acid and caffeic acid.

Cloning of COMT from Alfalfa (Medicago sativa)

Database searches revealed that the only COMT genes from Alfalfa which had been sequenced were all cDNAs, thus we had no idea as to whether the native COMT gene contained introns or not. Therefore we decided to both amplify the gene up from the Alfalfa plant, and to obtain a cDNA clone via reverse transcription.

The PCR amplification of COMT straight from the Alfalfa gene revealed there was no gene product present of ~1.1kb in size. From this we concluded the COMT gene must contain introns and proceeded to try and clone the gene via reverse transcription of COMT mature mRNA. Unfortunately for us we experienced some difficulty with producing COMT cDNA and decided to have COMT synthesised by GeneArt. The synthesised product was deposited in the Registry as BBa_I742107 (coding sequence alone) and BBa_I742109 (with a strong ribosome binding site).

Multi Host Plasmid pTG262

The plasmid we recieved had a multicloning site containing EcoRI, XbaI and PstI sites. To convert pTG262 into a BioBrick vector we inserted a BioBrick (Red Fluorescent Protein, BBa_I13521 or BBa_J04450, or lacZ', BBa_J33207) between the EcoRI and PstI sites. This simultaneously removed the intervening XbaI site, and introduced all four BioBrick restriction enzymes sites (EcoRI, XbaI, SpeI & PstI).

To create the BioBrick restriction sites, we inserted a total of three BioBricks:

Plac-RFP (BBa_J04450)(gives red transformants)
Ptet-RFP (BBa_I13521)(gives red transformants)
Plac-lacZ' (BBa_J33207) (gives blue transformants)

In order to test the plasmid in a variety of Gram negative bacteria, including Shewanella, Pseudomonas & Agrobacterium, we will transform the pTG262-Plac/RFP construct into our chosen host. This will enable easy detection, as all bacteria capable of retaining and expressing the vector will (we hope!) form easily detected red colonies, when grown on media containing lactose.

We have had success in transforming pTG262 into both E. coli and Bacillus subtilis using chloramphenicol selection (15 and 10 mg/l respectively). B. subtilis transformed with pTG262-BBa_I13521 did not give red colonies. This may be a promoter problem.

We have also attempted to transform Lactobacillus bulgaricus and Streptococcus thermophilus with these plasmids, but so far have not been successful, probably due to our lack of experience in working with these organisms. Since pTG262 is known to replicate in these bacteria, and since our modified versions can be used to transform Bacillus, we are (moderately) confident that these problems will be resolved.

Colonies of both Bacillus and E. coli containing pTG262 can be seen in Figure 3.

pTG262 BioBricks may be found in the registry under BBa_I742103 and BBa_I742123.

Limonene Biosynthesis

E. coli colonies containing Edinbrick1-Limonene synthase plasmids

We have recently received the limonene synthase gene from GeneArt and are working on ligating the construct into Edinbrick1 and pTG262 - hopefully there will be some gas chromatography results to report soon!

LIMS gene may be found in the registry at:

The Figure shows that the limonene synthase gene has succesfully been transformed into Edinbrick 1.

(shown by the presence of white colonies, which indicates that the lacZ' gene has been interrupted when the limonene synthase gene was ligated into the Edinbrick 1 vector)

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