IGEM:IMPERIAL/2006/LabCalendar/2006-9-8

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TO DO:
*Western Blot of Dr. Fray's plasmid containing aiiA
*Culture from plate from ligation from yesterday (preliminary predator cell with the new aiiA)
*Testing of J37015, J37016(for control), Acylase Activity & test a control too
*Make frozen stock of new T9002, J37036, J37024
*Control digest for J37036 - Has ligation worked?
*Have we ordered new Amp plates?
*Collect autoclaved flasks

  • JS: Just to point out, you will need to send to the registry the part inside bacteria and not the maxi-preped DNA!
  • CS: Yes. That's also one reason why we have been trying to make a frozen stock of all bacteria containing our parts that we ligated. How exactly do we need to send them to the registry?

Contents

Sequencing

  • Successfully sequenced J37020 - details can be found here
  • Sequenced J37025 (Includes J37023) - details here.
    • The sequencing revealed that the region purporting to aiiA was in fact a random sequence from a cloning vector. All the rest of the sequence was fine(the RBS and terminators). The incorrect aiiA sequence even had a degradation tag! This would suggest that the mistake is not due to us, but rather that the sequence sent to us was incorrect.This could well explain all the results for parts containing aiiA so far. However we want to make sure of this result. As a consequence we will sequence 5M which is the part from the well for confirmation.
  • The sequencing of J37015RS was not successful - this suggests that the riboswitch was not successfully incorporated into the construct because the primer used was the riboswitch primer. Perhaps re-sequencing using a plasmid primer would be more appropriate for definitive conformation. If we want to submit this then this should be done, however if not, then it is not necessary as we will not be using this construct anymore.
  • JS: If 5M turns out to be wrong, we should tell the registry about it. What is the back up plan? Didn't we get a new sequence from Dr. Fray? How is this working? VR: Funny, I had the impression that 'JS' was in charge of the back-up plan a long time ago ... ;)

LoxP PCR

  • The PCR fusion was a success - the resulting fragment will be ligated into a blunt end plasmid. Although we will not have time to construct J37015Cre, it would be good to put the part into one of our plasmids, and then do a few quick tests, for in theory the characterization of this part is not too time-consuming. Also it would be good if we were able to submit this part into the registry.

Protein Gel

  • Ran the protein gel for the new aiiA expression in Rosetta cells. It was going to be a 50:50 chance of whether or not we had a chance of getting some expression, as we do not at present know the orientation of the gene. (The plasmid has a T7 and Sp6 promoter, and only the T7 promoter can be expressed in Rosetta.)
  • This evening we looked at the gel. The initial viewing didn't appear to show any significant difference between those cells induced with IPTG and those not.
  • However the gel was still very stained and it should be looked at on Monday to confirm this
  • Sequencing the aiiA plasmid would enable us to determine whether or not we shoulod have any expression, but do we have a suitable primer??

J37036 and J37022 digest

  • J37036 was digested with several different enzymes - bands seem to be right, though as this part contains the incorrect J37025, it is slightly irrelevant......
  • J37022 only had one band - therefore it appears that it has not worked, though this may not matter now (see above)

Ampicillin plates

  • Not really something you would usually put into the lab notebook but..plates will be ready to pick up from the biochemistry basement at quarter to five (not before this time and no later than 5).

Miniprep

  • Alternative digest of new AiiA part (J37023) using Xbal and Spe1
  • Run on gel
  • Gel shows two bands corresponding to J37023 and T9 vector


Culturing

  • Set up "Bob" (J37024 AiiA-NEW/FRAY) for maxi and mini tomorrow.

Freezing Cells

  • Made a stock of frozen cells of:
    • T9002(d)
    • T9002(c)
    • J37036(a)
    • J37024(1)
    • J37024(2)
    • J37024(3)

Testing J37015

  • Following the revised J37015 Testing Protocol, this part was tested
  • J37015 taken out of shaker at 13:00 after morning dilution
  • Took readings every 10min. for 2hrs
  • J37016 was taken out of shaker after having been diluted into OD 0.1 in the morning (14:30)
    • OD: 1.184
  • For the control, 11 different concentrations of AHL were added to 1mL of cell culture of J37016 respectively
  • Using the diluted culture of J37016, inoculated J37016 cells with saved supernatant of J37015
  • Put in shaker at 16:25 (to be taken out after 4hrs)
  • Taken out of shaker at 20:05 to read OD and fluorescence


  • RESULTS OF TESTING:
    • Results from testing for concentrations of AHL (evening measurement):
      • An upward trend as time increases can be seen, however, data is quite messy
      • There is a peak of concentrations of AHL after about 80min. of measurements
      • At later time points, AHL concentrations seem to decrease again
(Data needs to be further processed - the results are going to be uploaded)

Testing Activity of Acylase

  • Following the Enzyme Activity Protocol, the acylase was tested using the J37016 culture as an assay for AHL
  • Additionally, a pH 7 buffer was added (25μL of 2M Tris HCl pH 7 to 1mL of cell culture)
  • Only concentrations F and G (see table in protocol) were tested. These are the highest concentrations of AHL, so if the enzyme will degrade these, it will also degrade lower concentrations of AHL.
  • As a control, J37016 was tested at the same time
  • Put in shaker at 15:45 (to be taken out after 4hrs)
  • Taken out of shaker at 20:05 to read OD and fluorescence

LB

  • 2L of LB has been made and put in for autoclaving. It should be done by the end of today.
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