Team:MichiganState/Notebook/HGT

Lab Notebook

General Anoxic HGT Biosensor Design

May

Week 1

05/20 - 05/25

Monday

  • Worked on DNA constructs for toxin/antitoxin system
Week 2

05/26 - 06/01

Tuesday

  • Reworked construct design, due to synthesis limitations
    • Repeating sequences could not be synthesized

Wednesday

  • Ordered ghoST construct

Thursday

  • Worked with anoxic group doing growth assays

Saturday

  • Volunteered at Recycle! East Lansing

June

Week 3

06/02 - 06/08

Monday

  • Made more 'P' media
  • Streaked LB + Kanamycin plate with E. Coli with the pAWP78 plasmid
  • Swabbed an isolated colony and inoculated in 5 mL of LB + Kan broth
  • Shook overnight at 30 ºC

Tuesday

  • Visited Granger Landfill Facilities
  • Did PCR of pAWP78 backbone with a temperature gradient of 55-75 ºC
  • Completed a plasmid extraction of the pAWP78 backbone
    • Total concentration was 65.65 ng/ul

Wednesday

  • Checked PCR of backbone with a gel
    • Did not get a strong band so will repeat with different parameters
Week 4

06/19 - 06/15

Tuesday

  • Did troubleshooting of PCR reaction of the pAWP78 backbone
    • Previous gel run based on PCR had too many bands
  • Ran two more PCR reactions with increased DNA
  • Ran two more PCR reactions with increased MgCl2
  • Ran gel based on PCR reactions
    • Increase in DNA did not work, but the increase in MgCl2 did
  • Made working 1 kb DNA ladder stock

Wednesday

  • Did PCR gel extraction of +1 mM MgCl2 PCR reactions for pAWP78 backbone
  • Performed a Gibson assembly with pAWP78 and ghoS
  • Transformed into S17 E. coli via electroporation
  • Transformed Gibson product into E Coli 10g via chemical transformation
  • Plated both transformations and left in 37 ºC overnight

Thursday

  • Checked plates and there was no growth
  • Redesigned primers to work correctly
Week 5

06/16 - 06/22

Monday

  • Got new primers for pAWP78 backbone
  • Ran pcr of pAWP78 and new primers
  • Ran a gel to confirm size-- no bands on gel

Tuesday

  • Ran a PCR with new primers with +2 mM MgCl2
  • Ran a new gel
  • Did a gel extraction
  • Did Gibson of extraction

Wednesday

  • Got colonies so did colony PCR and ran a gel
  • Cultured colonies from lanes 1,3 and 8

Thursday

  • Plasmid Purification of cultures from positive colony PCR yesterday
  • Sent for sequencing

Friday

  • Redid the colony PCR
Week 6

06/23 - 06/29

Monday

  • Streaked out JEB 1203 E. coli strain for UT Austin Burden Assay
  • Began growing E. coli strain with pCM80 for plasmid extraction

Tuesday

  • Plasmid extraction of the pCM80 plasmid
  • PCR of pCM80
  • Gel (no bands)
  • Inoculated 10mL of LB/Kan to with JEB 1203 to prepare to make competent cells

Wednesday

  • Began making competent cells
    • No growth because there was too much antibiotics
  • Ran 3 Different types of PCR for pCM80 (all on heat gradient 68-80)
    • Higher DNA
    • +1mM MgCl2
    • +2mM MgCl2
  • Ran a gel and still no bands
  • Trying PCR with lower concentration of primers tomorrow (less likely to dimerize)

Friday

  • Did a gel extraction

July

Week 7

06/30 - 07/06

Monday

  • Made 20mg/mL X-Gal stock
  • Made 100mM IPTG Stock
  • Made Blue-white Screening plates
  • Performed a Gibson assembly of pCM80 + ghoT
  • Transformed assembly into E. cloni 10g and plated on blue-white plates

Tuesday

  • Made LB/Cam Agar plates
  • Transformed 5 different JEB1203 Competent cells via electroporation with plasmids:
    • K608002
    • K608003
    • K608004
    • K608006
    • K608007
Week 8

07/07 - 07/13

Monday

  • Re-made blue-white plates because all colonies were white
  • Redid transformation of plasmid K608002 and plated on LB/CAM

Tuesday

  • Made 1mg/mL Cumate stock solution
  • Colonies were all white again on blue-white plates
  • Transformed pCM80 plasmid (no insert) into E. cloni 10g for negative control

Wednesday

  • Ran growth experiment of pCM80 + ghoT transformations
  • Added cumate solution for final concentration of 30ug/mL after 4 hours
    • Saw decrease in growth after cumate, but it picked up again after hour
    • Controls did not grow
  • Repeating Growth Experiment with adding cumate every hour on 15th

Thursday

  • CloneAmp PCR of pAWP78
  • Plated each tube from growth experiment on LB/TET, so I would have stocks

Friday

  • Ran gel for pcr pAWP78 pcr product
  • Gel extraction of bands pAWP78 PCR product
Week 9

07/14 - 07/20

Monday

  • Set up growth experiment to test the toxin efficiency when induced by cumate
  • Gibson Assembly of pAWP78 and ghoS

Tuesday

  • Transferred pCM80 (no insert) and pAWP78 (no insert) into S17-1 Cells
  • Made LB agar

Wednesday

  • PCR to amplify the ghoS insert
  • Ran gibson of pAWP78+ghoS with a 5:1 insert to backbone ratio
  • Gel extraction of ghoS insert
  • Colony PCR of pCM80+ghoT colonies

Thursday

  • Colony PCR of pAWP78 + ghoS

Friday

  • PCR of pAWP78
  • Plasmid extraction of pAWP78/ghoS bacteria
  • Plasmid extraction of pAWP78/ghoT bacteria
  • Sent ghoS and ghoT samples in for sequencing
Week 10

07/21 - 07/27

Monday

  • Helped other teams!

Tuesday

  • Electroporation of pAWP78/ghoS into S17-1 bacteria

Wednesday

  • Helped other teams!

Thursday

  • Colony PCR of pCM80 (no insert)
  • Mating protocol of S17-1 and 20Z

Friday

  • Team Break

August

Week 11

08/04 - 08/10

Monday

  • Made Cumate Stock solutions for ghoT growth curve
  • Started overnight cultures for the growth curve

Tuesday

  • Ran the ghoT + cumate growth curve

Wednesday

  • Met with Dr. Danny Ducat to discuss our progress on the burden assay collaboration with UT Austin

Thursday

  • Transforming pCM80 (no insert) and pAWP78 (no insert) into E. Cloni 10g
  • Transforming pCM80 +ghoT and pAWP78 + ghoS into E. Cloni 10g
  • Made LB/Tet plates
  • Ran burden assay
Week 12

08/11 - 08/17

Monday

  • Transforming DAP dependent WM3064 with pAWP78/ghoS and pCM80/ghoT
  • Transforming BBa_1608002 into E. cloni 10g
  • Made DAP agar

Tuesday

  • Transforming DAP dependent WM3064 with pAWP78/ghoS and pCM80/ghoT again
Week 13

08/18 - 08/24

Monday

  • Making overnight cultures of E. cloni 10g + pCM80/pAWP78
  • Growing WM3064 ghoST, WM3064 pAWP78/pCM80, and E. cloni K608007 for gene transfer assay

Tuesday

  • Ran horizontal gene transfer assay
  • Ran ghoST and WM3064 pCM80/pAWP78 growth assay on plate reader

Thursday

  • Performed Plate counts for the Horizontal Gene transfer assay
Week 14

08/25 - 08/31

Tuesday

  • Met with team and discussed what to do next with the burden assay

September

Week 15

09/01 - 09/07

No activity this week!

Week 16

09/08 - 09/14

Sunday (09/08)

  • PCR of the ghoS construct with MtaC primers to replace the pMoA promoter

Tuesday

  • Transformation of the 12 Cam resistant plasmids into JEB1203 for the burden assay
  • Transformation of the mtac/ghoS into E. cloni 10g

Wednesday

  • Colony PCR on the mtac/ghoS transformations from yesterday

Thursday

  • Redoing 11/12 of the CAM resistance transformations from 9/10
  • Miniprepped transformations that got at least 1 colony
Week 17

09/15 - 09/21

Tuesday

  • Transformation of CAM resistant plasmids that didn’t work again
  • Made M9CA Minimal Media
  • dpnI digest and pcr cleanup of the mtac/ghoS PCR product

Thursday

  • Gibson Assembly of the mtac/ghoS PCR
  • Plasmid Purification of Burden assay plasmids
Week 18

09/22 - 09/28

Tuesday

  • Transformation of the mtac/ghoS dpnI digest into E. cloni 10g
  • Transformation of Burden assay plasmids from the minipreps
  • Made LB/Amp agar plates for the remaining Amp resistant burden assay plasmids

October

Week 18

09/29 - 10/05

Week 19

10/07 - 10/11

Week 20

10/14 - 10/18