Team:University College London/LabBook/Week10

Tuesday 14.8.12

Aim - Repeat experiment 9.2: We think there might have been a confusion in preparing the samples for the PCR because we did not obtain any bands on the gel for irrE or Laccase.

Method

Step 1 - Setting up PCR tubes: The table below gives the identity of the primers used for each reaction. It indicates the samples that were set up for the repeat of Expt 9.2, as was done in the first attempt of Expt 9.2.

Results: The image below shows a 1% Agarose Gel of an Analytical Restriction Enzyme Digest for Expt 9.2, with a 1000bp ladder. Again we have not obtained any bands. In Lanes 1 and 3 we expected a product corresponding to the size of the irrE gene (1974bp) as indicated by A and C respectively. C would be marginally larger because it also contains the standard BioBrick prefix and suffix, but this different would not be noticeable on a gel. In Lane 2 and 4 we would expect products corresponding to the size of the Laccase gene (1554bp), as shown by B and D. Again we would expect D to be a slightly larger product because the primers were designed to include the prefix and suffix, but this is not a difference we would expect to detect. The strong patches of white demonstrate to us that our DNA template is at a high concentration, and should be diluted before we attempt any repeats. The lack of any products suggest we need to reconsider the protocol. Revising the annealing temperature or designing new primers will have to be considered.

Conclusion: We will revise the protocol to see if we can detect any bands.

Wednesday 15.08.12

Aim - Repeat the PCR again to optimise results: As an improvement we are using: - Phusion polymerase - Diluting the irrE DNA 10-fold, due to the excessive dose on DNA shown on the gel from yesterday. - Changing the annealing temperature from 55C to 57C

Method

Step 1 - Setting up PCR tubes: The polymerase used was Phusion, and we had two template DNAs – irrE and Laccase.

Step 2 – PCR Program: An annealing temperature of 57oC was used instead of the standard 55oC.

Results: The image below shows a 1% Agarose Gel of an Analytical Restriction Enzyme Digest for Expt 9.2, with a 1000bp ladder. Again we have not obtained any bands. A and C correspond to the bands expected for Laccase, and B and D for bands expected for irrE. The final two columns are negative controls for which no product was expected. The reason for the failure of this gel is unclear.

Monday 13.8.12

Aim - Check the results of the weekend culture of plates: At the end of last week we set up Expt 9.4, to assess whether poor activity of our antibiotics was a cause of the poor results we get after colony picking. We proposed that our antibiotics may be weak, allowing opportunistic colonies to grow on agar. Therefore we set up a series of agar plates with various antibiotics and controls, to see if we could prove that bacteria without resistance is capable of growing on antibiotic inoculated agar. If this is the case, we know our antibiotic is weak.

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Monday 13.8.12

Aims – To test the growth of marine bacteria in different media: This is to optimise the conditions of growth for the bacteria. A test for the marine bacteria will be compared to control E.coli, and the difference in growth measured using a spectrometer. The two media being tested are LB media (typically used for E.coli) and marine media. We expect to have greater optical density of indoliflex in marine medium & greater optical density for W3110 in LB.

Methods

Step 2 – Inoculating Colonies into a Selective Broth: The table below indicates the volume of broth and the concentration of antibiotic required for each BioBrick. Each cell type will be inoculated into both types of media.

Tuesday 14.8.12

Aim: To measure the optical density of bacterium after overnight culture in different media.

Method?

Results: The table below summarises the results of OD600 measurement. As expected, all three samples of W3100 grown in LB media have a higher optical density (and therefore growth) than the single sample of W3100 grown in marine media. The marine bacterium Oceanibulbus indoliflex also gave us our expected results, with the four samples grown in marine media giving higher optical density (and therefore growth) than the two samples grown in LB media.

Conclusions

This demonstrates the better suitability of marine media for the marine bacterium. Glycerol stocks were made of the W3100 Sample 2 cultured in LB media, and of the Oceanibulbus indoliflex Sample 3 culture in marine media, as these had the highest density. The W3100 sample will be used for competent cell preparation.