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From 2011.igem.org

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Module 2: Auxin Xpress

Auxin, or Indole 3-acetic acid (IAA), is a plant growth hormone which is produced by several soil bacteria. We have taken the genes encoding the IAA-producing pathway from Pseudomonas savastanoi and expressed them in Escherichia coli. Following chemotaxis towards the roots and uptake by the Phyto Route module, IAA expression will promote root growth with the aim of improving soil stability.

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Assembly

We wish to build a single expression plasmid that can express IaaH and IaaM. While this task can be summarised in one sentence its execution is not as short. The first problem lies in the size of these two enzymes which both exceed 1kbp making their synthesis a problem. We therefore created a new standard for biobrick assembly to tackle this issue. We broke up these large sequences into four fragments that were ordered at the end of week 3. In preparation for the arrival of these fragments (circa 8-10 days) we started to transform our cells with the pVEg+pSB1C3 backbone constructs in order to make enough genetic material for a gibson assembly reaction. This chapter will describe our struggles and successes throughout this grueling and yet rewarding process.

29th of July

In preparation for the arrival of our sequences, we transformed cells with pSB1C3 containing BBa_K398500 and J23100 promoter (cell line 6). We also transformed the cells with part BBa_K316001 (cell line 7) and part BBa_K316005 (cell line 8).

Colonies from cell lines 6, 7, and 8 were mini-prepped and restriction digested with PstI and EcoRI to verify their size (pSB1C3 with the appropriate promoter).

Gel 1&2. Restriction digest of three backbone vectors with EcoRI(E) and PstI(P) to confirm backbone length. Gel1: Lane1-1 kb DNA ladder Lane 2-6a cut with E;Lane 3-6a cut with P; Lane 4-6a cut with E+P;Lane 6-6b cut with E; Lane 7- 6b cut with P; Lane 8- 6b cut with E+P; Lane 11- 7a cut with E; Lane 12- 7a cut with P; Lane 13- 7a cut with E+P; Lane 15- 7b cut with E; Lane 16- 7b cut with P; Lane 17- 7b cut with E+P. Gel 2: Lane 1 - 1kb ladder; lane 2 - 8a cut with E; lane 3- 8a cut with P; lane 4- 8a cut with E+P; lane 6- 8b cut with E; lane 7- 8b cut with P; lane 8 - 8b cut with E+P.

4th of August

Today we redid the PCR of samples 6, 7, and 8 but with a temperature gradient to improve primer annealing.... and it was a success! So tomorrow we can run the rest of the Dpn1 digested DNA on a gel and gel purify it, then the vectors are ready to be used for DNA assembly once our genes arrive!

Gels 3&4: Temperature gradient PCR of desired backbone with promoter and terminator out of plasmids. Gel 3:Lane 1- 1kb DNA ladder; lanes 2 to 7 - PCR of vector 6 from 57.1°C to 62.6°C; lanes 9 to 15 - PCR of vector 7 from 57.1°C to 62.6°C. Gel 4: lanes 2 to 7 - PCR of vector 8 from 57.1°C to 62.6°C

The backbone sequences have also returned. All of the samples are in order except a one base pair mutation in sample 8 within the pVEg promoter. For now, we are going to amplify it anyways in the hope that the one base pair mutation is just an error that occured during sequencing. Either way, sample 8 is a back-up of sample 7 so there should be no problems either way.

8th of August

Today we ran a gel of the PCRd backbone DNA extracted from the previous gel to make sure that the DNA was pure. The gel results were succesful. We also transformed cells with the pure DNA to check that the Dpn1 digestion worked properly.

Gel 5: Gel extracted backbone vector DNA run on a gel to confirm purity. Lane 1- 1 kb DNA ladder; Lane 2- vector 6a; Lane 3- vector 6b; Lane 4- vector 7a; Lane 5- vector 7b; Lane 6- vector 8a; Lane 7- vector 8b.

9th of August

Transformations of auxin fragment 1 (20) and auxin fragment 4 (24) were successful. We obtained plenty of colonies to choose from on both the ampicillin and kanamycin plates. Also, the DpnI digest transformations created bacteria that had no resistance to chloramphenicol.

However, sample 8 has to be repeated because in the "rest" plates there were 1 or 2 colonies on each.

10th of August

Today we attempted to perform a midi-prep on the DNA fragments that had arrived from Germany. The experiment failed and we were not able to obtain a decent yield of DNA. Oh well, got to try again.

12th of August

After the failed results and the rather lethargic week we attempted to get our minds back to gear. The visit to Syngenta the day before was a moment of respite that allowed us to perform two mini-preps that worked. However, there was an issue when we digested our plasmid (pCR2.1) with MlyI. The genes had been placed in a plasmid that contains multiple MlyI sites which would make the gel extraction more difficult. We used Serialcloner to make a virtual cut and then used the predicted image to guide us. In the end we obtained a band for 20b and 24a in the right location (or so we hope!).

Gel 6: gel of MlyI restriction digest of the synthesised genes. Lane 1-Marker; Lane 2-20a; Lane 3-20b; Lane 4-22a; Lane 5-22b; Lane 6-23a; Lane 7-23b; Lane 8-24a.

15th of August

We gel extracted several gene fragemtns that were transformed yesterday.

16th of August

The transformations we did the previous day were a success and the last two remaining fragments were also mini-prepped. We will attempt the Gibson assembly. If all goes well, we'll have E. coli excreting auxin by Friday!

19th of August

Gel 7: Gel extracted DNA fragments for assembly of auxin expressing plasmid. Lane 1&2 - Auxin fragment 1; Lane 3 - Auxin fragment 4; Lane 4&5 - Auxin fragment 3; Lane 6&7 - Auxin fragment 2; Lane 8&9 - pVEG backbone vector

22nd of August

Today we obtained some disappointing results. It seems like the vector is just religating during the Gibson reaction. Maybe the sequences of the two ends of the vectors are too homologous for Gibson to work. Either way, we will be attempting CPEC today and hopefully we will obtain some bands that we can purify and transform bacteria with.

CPEC Assembly

We assembled our four auxin fragments along with the promoter containing backbone using CPEC. CPEC (Circular Polymerase Extension Cloning) is a primer-independent PCR assembly technique which relies on overlaping sequences between each part to be assembled. With a denaturing step, the double stranded DNA is melted, allowing compatible single stranded ends of each part to join. For this reason it is essential that the parts are designed with homologous ends (the fragments we used were designed with 50 bp overlaps). The annealed overlapping ends then serve as primers for polymerase extension to join the parts into a seemless construct.

We were firsty time lucky with CPEC! We quickly verified the assembly by doing a PCR of the CPEC assembly with our standard sequencing primers which anneal to the promoter and terminator of the pC13b backbone, so we would expect it to PCR the insert which should be around 4 kb if it worked. We also transformed cells with the assembled construct and performed a colony PCR. The PCR products were run on an analytical agarose gel (shown below) and all of the bands corresponded to the expected sizes.

DNA was mini-prepped from colonies and sequenced by Eurofins. The sequences came back positive so we could move on and start characterizing the auxin construct.

Gel 8: The first two lanes show that CPEC assembly of four auxin fragments at ~1kb each in a backbone of about 2kb. Lane one contains the assembled construct at ~6 kb and lane 2 contains the negative control assembly of backbone vector with no insert at ~2kb. The following two lanes show the analytical PCR of the CPEC assembled product with standard biobrick primers to PCR our the assembled auxin fragments. The first well shows the auxin assembly at ~4kb and the second (negative control) shows no PCR product because no insert is present. Gel 9: Colony PCR with standard biobrick primers of CPEC assembled auxin fragments showing the desired assembly size of about 4 kb. Gel 10: Colony PCR of negative control colonies (backbone vector 8 only and no insert) and positive control colony PCR of the same vector 8 but the entire plasmid. This result shows that the DpnI digest of PCRd backbone vector 8 was not completely efficient as some complete plasmid remains, but this residual amount did not hinder assembly.

25th of August

A restriction digest of the mini-prepped auxin assembled constructs with EcoRI and PstI show clearly the assembled auxin insert drop out of the backbone vector.

Gel 11: Restriction digest of auxin construct with EcoRI, PstI and both for three miniprepped colonies grown from transformed E. coli.