Latest revision as of 01:21, 17 October 2014

Exeter | ERASE

Digestion, Ligation, Transformation and Purification (Days 1-10)

Summary:

Our parts BBa_K1398001, BBa_K1398003, BBa_K1398004 and BBa_K1398007 were successfully transformed into the iGEM vector pUC19 and were successfully transformed into DH5α and TOP10 E.coli strains for our future experiments.
Promoters BBa_J23100-BBa_J23119 (A+C), the RBS BBa_B0031-34 and the double terminator BBa_B0015 were all successfully transformed into competent cells for future experiments.

Introduction

Once we had designed our constructs and drafted out a plan of the goals we aimed to achieve, we began preparative work in the laboratory.

One of our early, what we termed ‘foundational’ ideas, revolved around characterising the expression profiles of a vast number of different promoter and RBS combinations to generate a map of various expression rates that could be utilised to fit certain needs. We aimed to build this information into a ‘machine learning’ programme which could predict possible combinations based on gene sequences. However, while we had hoped to test over 400 combinations of promoters, RBS and GFP/RFP using a liquid handling robot a key issue we were mostly unable to overcome was the digestion and ligation of individual parts as small as promoters and RBS onto plasmid backbones.

As a result many of our earlier experiments involve the transformation of a large number of promoters and RBS into competent cells. However, this phase also involves the important transformations of our gene constructs (designed by us but supplied by https://www.dna20.com/ ) into the iGEM vector, essential for our later experiments and for submission to iGEM.

Written Account

'''Day 3 (01/07/14):''' We took the RBS BBa_B0030-34 and the double terminator BBa_B0015 and followed steps 1-12 of the transformation protocol provided by iGEM http://parts.igem.org/Help:Protocols/Transformation. The day ended with RBS BBa_B0030 and the positive control on separate LB 20μl and LB 200μl ampicillin (AMP) resistant plates and the RBS BBa_B0031-34 and terminator BBa_B0015 on LB 20μl and LB 200μl chloramphenicol (CAM) resistant plates being cultured overnight.

'''Day 4 (02/07/14):''' On day four we found that while the RBS BBa_B0031-33 and double terminator BBa_B0015 were successfully cultured on plates, the two RBS BBa_B0030 and BBa_B0034, along with both controls, returned no colonies on either the 20μl or LB 200μl plates and were unsuccessful. Thus we repeated the transformations of RBS BBa_B0030 and BBa_B0034, this time culturing 30 on AMP and 34 on CAM. We also performed the transformation of the promoters BBa_J23100-05 and the two promoters BBa_J23119 A and BBa_J23119 C. In both cases we again followed steps 1-12 of the transformation protocol provided by iGEM http://parts.igem.org/Help:Protocols/Transformation (See pages 4 and 5 hyperlink).

'''Day 5 (03/07/14):''' All promoters transformed on day 4 were transformed successfully; glycerol and mini-preps of the RBS and terminator were prepared and the optical density and growth rate of one of our E. coli strains TOP10 was measured in order to prepare competent cells at standard concentration. (See pages 7 and 8 and E. coli stress testing for more information on growth rates.) All of the promoters from day 4 (BBa_J23100-05) grew up on both the LB 20μl and LB 200μl AMP resistant plates. Promoter BBa_J23119 A grew only on the LB 200μl AMP plate and Promoter BBa_J23119 C grew successfully on both the LB 20μl and LB 200μl CAM resistant plates. All of these parts had single colonies picked towards the end of the day and placed in falcon tubes of liquid LB agar for overnight incubation at 37oC in the shaking incubator. We prepared the glycerol stocks, by adding 0.5ml of the culture of each part to 0.5ml of 80% sterile glycerol in labelled micro-centrifuge tubes. These were then quickly vortexed, dropped into liquid nitrogen and then stored in the -80oC freezer for future use. To purify the plasmid DNA we utilised a Thermofisher Scientific GeneJET Plasmid Miniprep Kit (http://www.thermoscientificbio.com/nucleic-acid-purification/genejet-plasmid-miniprep-kit/) The protocol for which is available: http://www.thermoscientificbio.com/uploadedFiles/Resources/protocol-K0502-K0503-GeneJET-Plasmid-Miniprep-Kit.pdf We had two alterations to this protocol. Namely that during step 3 (Washing the column) we performed centrifugation for 5 minutes rather than for 1 minute to ensure residual solution had been removed, and during step 4 our supervisors suggested that we used 50μl of de-ionised water rather than elution buffer.

'''Day 6 (04/07/14):''' On this day we prepared a large number of transformations of promoters BBa_J23106-08 on LB 20μl and LB 200μl AMP resistant spread plates, a repeat transformation of RBS 30 and transformations of our construct parts which had arrived synthesised from DNA 2.0 (https://www.dna20.com/ ) (See page 9) . Once again, we followed steps 1-12 of the transformation protocol provided by iGEM http://parts.igem.org/Help:Protocols/Transformation.

'''Day 7 (07/07/14):''' This was the day in which we attempted our first restriction digest and ligation of our purified parts to the iGEM vector to form the recombinant plasmids of our final constructs suitable for submission to iGEM (See page 10 ). It was important to balance the concentrations of DNA to backbone to ensure a successful and efficient digestion and ligation of our part to the backbone. Therefore, the restriction digest protocol that we followed (provided by iGEM http://parts.igem.org/Help:Protocols/Restriction_Digest ) required 250ng of each construct’s DNA to a known concentration of backbone. In order to discover the volume of our DNA solution required for this 250ng mass we utilised a Qubit® DNA quantification machine. Using two standards we generated a standard curve and then used this to calculate the concentrations of DNA in our construct samples and therefore the relative volumes of each part required. With this information we then made up the volume of DNA in each tube to a total volume of 16ul with distilled water and followed steps 1-9 from the iGEM protocol (http://parts.igem.org/Help:Protocols/Restriction_Digest ). This resulted in each of the parts being digested from their original backbones and the intended backbone target being linearized. We then followed the ligation protocol outlined by iGEM (http://parts.igem.org/Help:Protocols/Ligation ) and then transformed the resulting recombinant plasmids into TOP10 E. coli competent cells once again following steps 1-12 of the transformation protocol provided by iGEM http://parts.igem.org/Help:Protocols/Transformation. This resulted in our parts on the PSB1C3 backbone being spread onto LB 20μl and LB 200μl AMP resistant spread plates and cultured overnight. Lastly, promoters BBa_J23106 and RBS 30 were successful on both LB 20μl and LB 200μl AMP resistant plates. However, BBa_J23107 and BBa_J23108 were unsuccessful on both, leading us to believe that a different antibiotic resistance was required.

'''Day 8 (08/07/14):''' Our attempts at producing colonies containing our constructs using TOP10 competent cells failed at both concentrations and we performed transformations of promoters BBa_J23107-12. Additionally, we generated 20 LB AMP plates and 18 LB CAM plates, which we stored in the cold room for future use (See pages 11 and 12). We reasoned this failure was because PSB1C3 was a smaller plasmid backbone than we calculated during the transformation efficiency. We repeated the second attempt of our experiment with three main alterations:

1. We altered the calculated values for the amount of buffer and construct based on a new assumed backbone size, 2. We decided to grow cultures containing 1:1 and 3:1 ratios of backbone to construct to enhance chances of successful ligation, 3. We also decided that these cultures would be in the two E.coli strains DH5α and TOP10, once again in order to maximize our chances of success.

'''Day 9 (09/07/14):''' This day was a milestone for the team in that we were successful in the addition of our constructs BBa_K1398001, BBa_K1398003, BBa_K1398004 and BBa_K1398007 into both DH5α and TOP10 for our future experiments and in the addition of these constructs into the iGEM vector PUC19. The later was then submitted for genetic sequencing to ensure that our construct had been transformed into the competent cells correctly (See page 13 ). Promoters BBa_J23107-12 were all successfully expressed and we then performed transformations of the promoters BBa_J23113-18 following steps 1-12 of the transformation protocol provided by iGEM http://parts.igem.org/Help:Protocols/Transformation on AMP plates.

'''Day 10 (10/08/14):''' All of the promoters BBa_J23113-18, apart from BBa_J23116, were successfully transformed and grown on both LB 20μl and LB 200μl AMP resistant spread plates (See page 13 ). We discovered that BBa_J23116 had failed to be transformed due to issues with the initial extraction from the well on the iGEM plate.