Team:Cambridge/Lab book/Week 12

Monday (10/09/12)

PCR of biobrick vector DNA

• Attempted to run PCR of the backbone for the biobricks once more. Settings: Annealing temperature: 56°C, elongation time: 30secs.

• After running on gel, saw that while the -8 vector amplification appears to be producing some correctly sized (~2kb) bands, the other two only seem to be forming many primer dimers. Analysis of the sequence of the prefix and suffix revealed a CG palindrome that appears to be causing self annealing. Inserts are not affected, as they have the palindrome at the 5' end of the primer.

• Will retry at higher temperatures tomorrow.

Gibson assembly of biobricks

• Attempted Gibson assembly for the -8 magnesium riboswitch biobrick with the backbone fragments produced earlier today and the insert fragments produced yesterday.

Transformation of e.coli with Gibson products

• E.coli cells transformed with Gibson products produced earlier today.

• Transformants plated out on 25μ/ml chloramphenicol plates.

Restriction digest of magnesium riboswitch construct plasmids

• Colonies grown up from colonies produced from Gibson products made on Check!!! were miniprepped and plasmid DNA extracted.

• Restriction digest performed with Sal1 and BamH1. Expected fragment sizes: 4900bp and 4500bp. If plasmid lacks insert: 4900bp and 3950bp. These two possibilities should be distinguishable.

• Correct banding pattern seen for all colonies grown up, with both -8 and +8 construct. Shall verify identity further with colony PCR.

Tuesday (11/09/12)

PCR of biobrick backbone

• Retried PCR of backbone for +8 magnesium riboswitch biobrick and fluoride biobrick at 60 °C and 64 °C.

• Verification gel showed that no bands of the appropriate size came out. Once more, only primer dimers produced.

• Production of primer dimers at these temperatures causes us to doubt that a successful PCR will be carried out at these temperatures. We may have to switch to ordinary ligation to construct our biobricks for submission.

Colony PCR of biobrick containing colonies

• Gibson assembly of biobrick plasmid yesterday produced a few colonies. These were tested with the standard sequencing primers to check if an insert of the correct size had been introduced into the bacteria.

• Gel showed that fragments of the appropriate size were produced for one of the -8 biobricks and one of the -8 colonies. These will be grown up, miniprepped and sent for sequencing.

Wednesday (12/09/12)

Ratiometrica - lux

• DNA 2.0 construct arrived

• Colonies restreaked and put into 30 °C incubator.

• Only one set of colonies appears to be fluorescing, which may be of concern.

Thursday (13/09/12)

Magnesium Riboswitch Plate-Reader Assay

• Plate reader assay set up with magnesium riboswitch construct made over the last few days.

• Mg2+ concentrations used:

• 5μM, 10μM, 20μM, 50μM, 100μM, 200μM, 500μM, 1mM, 2mM, 5mM, 10mM, 20mM

• IPTG concentrations used:

• 0.05mM, 0.1mM, 0.2mM, 0.5mM, 1mM, 2mM, 5mM, 10mM

• Magnesium free medium B used as growth medium

• Results: fluorescence curves are constant, except for a distinct dip during the exponential phase of bacterial growth. After looking at various cultures under the fluorescence microscope and discussing the results with our advisors, we have decided that this is because the amino acids in the medium are fluorescing. Our bacteria are then degrading these, causing the dip observed. We will switch to M9 minimal medium for our assays in the future, as this does not autofluoresce.

Friday (14/09/12)

PCR of α and β fragments for ratiometrica

• Although ratiometrica appears to have produced successful colonies, we would still like some extra fragments in case we need to repeat our construction.

• Previous successful gel extractions of α and β used as template for PCR.

• α has successfuly been amplified, but &beta produced band of the wrong size.

Gel extraction of α

• α successfully extracted from gel.

Saturday (15/09/12)

Ratiometrica - lux

• Attempted digestion of DNA 2.0 pure plasmid with EcoRI and SpeI, expected fragments 2648bp and 8738bp.
• Parallel digestion of pSB1C3 backbone ready for ligation
• 8738 band extracted, was a little weak
• 8738 band and digested backbone ligated together
• Chemically competent E. coli used for transformation
• Plated on LB Agar with 25μg/ml Chlor

Sunday (16/09/12)

Ratiometrica - lux

• Picked colonies from ligation attempt from yesterday, some red colonies on plates, these were avoided with one exception as a control.
• Picked colonies grown in LB with 25μg/ml Chlor