Team:Michigan/Pili October

From 2010.igem.org

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==10/2/2010==
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''Kevin''
 +
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Prepared tubes with K12 and ΔFim (Both w/insert) for an agglutination assay. We added algae and arabinose at an OD600 of 0.8 and then let the tubes grow
 +
 +
==10/3/2010==
 +
''Kevin''
 +
 +
We were able to observe pellets in the tubes containing ΔFimB w/insert. This is exciting, but there are several things to note:
 +
*By looking at the pellets under a microscope, we were able to see many flocs, some of which were a combination of E coli and algae, and some of which were pure E coli. This is a promising result.
 +
*Both induced and uninduced tubes of ΔFimB formed flocs, however the arabinose induced tube had a much larger floc. This is possible evidence of leaky expression.
 +
*None of the K12 tubes formed flocs.
 +
 +
We are planning to run a more quantitative assay later this week.
 +
 +
==10/7/2010==
 +
''Kevin''
 +
 +
Ran flocculation assay.
 +
#Prepared O/N cultures of K12 and ΔFimB, both with the FimB plasmid.
 +
#Created 5 tubes: LB, LB+ΔFimB (both uninduced and induced), LB+K12 (both uninduced and induced)
 +
#*Used 5 ml of cell culture due to low algae culture available
 +
#Added 100 ul arabinose during exponential phase
 +
#Added 5 ml algae to every tube
 +
#Shake for 10 min
 +
#Take 200 ul samples and measure OD600.
 +
 +
[[Image:ODassay10_8.png|800px]]
 +
 +
This graph illustrates the results. We can see a decline in the OD600 for all species, but there is no clear distinction between our control tube (LB+Algae) and the other tubes containing E. coli.
 +
 +
Suggested Modifications:
 +
*This procedure was originally developed to measure a fast-acting chemical flocculant. Clearly, the time-scale will have to be changed to adjust for the slower E. coli.
 +
*In between measurements, the tubes were allowed to sit at room temperature. It may be more beneficial to put the tubes in the incubator shaker, and take measurements less frequently.
 +
*If we wait 2-3 hrs for the arabinose to fully induce and then start taking measurements, we should get better results.
 +
 +
Marc ran another qualitative assay, with positive results.
 +
 +
[[Image:Floc_tubes10_8.jpg|400px]]
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 +
==10/13/2010==
 +
''Kevin''
 +
 +
Ran PCR of FimB in preparation of making our final iGEM BioBrick standard part.
 +
 +
==10/15/2010==
 +
''Kevin''
 +
 +
Ran gel of PCR products from 10/13. It appears that all of our wells contain our intended product, however 3 wells appear to contain unexpected products as well.
 +
 +
[[Image:Gel10_15_10.png|400px]]
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[[#top|Back to top]]
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==10/18/2010==
 +
''Kevin''
 +
 +
PCR purified the FimB PCR product, using the PCR purification kit in 1230 ERB. Started o/n cultures of ΔFimB and K12 in preparation of another OD assay.
 +
 +
Performed nanodrop to determine concentration of products.
 +
*I had two separate tubes of product due to the PCR purification.
 +
Results:
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!
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! FimB Digest 1
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! FimB Digest 2
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|-
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| 260/280
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| 1.85
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| 1.86
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|-
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| 260/230
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| 2.10
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| 2.02
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|-
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| ng/uL
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| 39.2
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| 36.0
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|}
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Performed digest of FimB, using EcoR1 and Pst1. Ran the digest alongside VP130 and Flu.
 +
 +
Performed ligation of FimB into pSB1C3, the standard iGEM plasmid backbone. Used the quick ligation kit.
 +
 +
Performed transformation of FimB into competent cells. Made plates of 1:1 and 1:3 dilutions.

Revision as of 05:23, 19 October 2010


Michigan Header




Sunday Monday Tuesday Wednesday Thursday Friday Saturday
Week 14 - - - - - - 10/2/2010
Week 15 10/3/2010 - - - 10/7/2010 - -
Week 16 - - - 10/13/2010 - 10/15/2010 -
Week 17 - 10/18/2010 - - - - -

10/2/2010

Kevin

Prepared tubes with K12 and ΔFim (Both w/insert) for an agglutination assay. We added algae and arabinose at an OD600 of 0.8 and then let the tubes grow

10/3/2010

Kevin

We were able to observe pellets in the tubes containing ΔFimB w/insert. This is exciting, but there are several things to note:

  • By looking at the pellets under a microscope, we were able to see many flocs, some of which were a combination of E coli and algae, and some of which were pure E coli. This is a promising result.
  • Both induced and uninduced tubes of ΔFimB formed flocs, however the arabinose induced tube had a much larger floc. This is possible evidence of leaky expression.
  • None of the K12 tubes formed flocs.

We are planning to run a more quantitative assay later this week.

10/7/2010

Kevin

Ran flocculation assay.

  1. Prepared O/N cultures of K12 and ΔFimB, both with the FimB plasmid.
  2. Created 5 tubes: LB, LB+ΔFimB (both uninduced and induced), LB+K12 (both uninduced and induced)
    • Used 5 ml of cell culture due to low algae culture available
  3. Added 100 ul arabinose during exponential phase
  4. Added 5 ml algae to every tube
  5. Shake for 10 min
  6. Take 200 ul samples and measure OD600.

ODassay10 8.png

This graph illustrates the results. We can see a decline in the OD600 for all species, but there is no clear distinction between our control tube (LB+Algae) and the other tubes containing E. coli.

Suggested Modifications:

  • This procedure was originally developed to measure a fast-acting chemical flocculant. Clearly, the time-scale will have to be changed to adjust for the slower E. coli.
  • In between measurements, the tubes were allowed to sit at room temperature. It may be more beneficial to put the tubes in the incubator shaker, and take measurements less frequently.
  • If we wait 2-3 hrs for the arabinose to fully induce and then start taking measurements, we should get better results.

Marc ran another qualitative assay, with positive results.

Floc tubes10 8.jpg

10/13/2010

Kevin

Ran PCR of FimB in preparation of making our final iGEM BioBrick standard part.

10/15/2010

Kevin

Ran gel of PCR products from 10/13. It appears that all of our wells contain our intended product, however 3 wells appear to contain unexpected products as well.

Gel10 15 10.png

Back to top

10/18/2010

Kevin

PCR purified the FimB PCR product, using the PCR purification kit in 1230 ERB. Started o/n cultures of ΔFimB and K12 in preparation of another OD assay.

Performed nanodrop to determine concentration of products.

  • I had two separate tubes of product due to the PCR purification.

Results:

FimB Digest 1 FimB Digest 2
260/280 1.85 1.86
260/230 2.10 2.02
ng/uL 39.2 36.0

Performed digest of FimB, using EcoR1 and Pst1. Ran the digest alongside VP130 and Flu.

Performed ligation of FimB into pSB1C3, the standard iGEM plasmid backbone. Used the quick ligation kit.

Performed transformation of FimB into competent cells. Made plates of 1:1 and 1:3 dilutions.

Retrieved from "http://2010.igem.org/Team:Michigan/Pili_October"