Team:MichiganState/Notebook/Anoxic

Lab Notebook

General Anoxic HGT Biosensor Design

May

Week 1

05/19 - 05/25

Monday

  • Completed Safety Training
  • Drafted DNA constructs

Tuesday

  • Continued work with DNA constructs
  • Explored other teams wiki designs

Wednesday

  • Discussed tagging DNA constructs with histidine
  • Created Social Media Accounts
  • Began drafting survey

Thursday

  • Met with a graphic design consultant the wiki page
  • Began learning and practicing HTML for wiki
  • Began drafting survey

Friday

  • Continued troubleshooting constructs
  • Worked on wiki interface
Week 2

05/26 - 06/01

Tuesday

  • Ordered Constructs!! Seven inserts ordered from IDT
  • Trained on sterile technique
  • Prepared 'P' Media

Thursday

  • Inoculated culture of Methylomicrobium alcalphilum 20Z in 1 mL of 'P' Media
  • Did first trial run of growth assay
    • Used 9 tubes with oxic methanol, "anoxic" methane, and "anoxic" methanol
    • Each experimental group was done in triplicate
    • The "anoxic" groups, however, were not done in the anoxic chamber
  • Monitored and recorded optical densities (OD)

Friday

  • Continued taking OD for the cultures from the previous day

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 117.45 ng/ul

Wednesday

  • Checked PCR of backbone with a gel
    • Did not get a strong band so will repeat with different parameters
  • Set up another growth assay
    • This time with 12 tubes: oxic methane, oxic methanol, anoxic methane, anoxic methanol
    • The anoxic experimental groups were done in the anaerobic chamber for the first time
  • Recorded the OD at hour 0 and shook overnight at 30 ºC

Thursday

  • Continued taking optical density for growth assay
  • Did another PCR of backbone with a DMSO gradient and a temperature gradient
    • The DMSO gradient was 3%, 6%, and 9%
    • The temperature gradient was adjusted to 65-80 ºC
  • Ran a gel of the PCR products

Friday

  • Prepared more "P" media
  • Weekly meeting to communicate progress and discuss future work
Week 4

06/09 - 06/15

Monday

  • Began set up to repeat growth assay again by moving materials to anoxic chamber
  • Completed Gibson Assembly in a 2:1 ratio of inserts to pAWP78 backbone
    • Did three separate reactions for each of our three constructs

Tuesday

  • Using the Gibson assemblies, did transformation via heat shock
  • Plated the transformed cells and grew overnight
  • Set up another growth assay
    • This time with 18 tubes: oxic methane, oxic methanol, anoxic methane, anoxic methanol, microaerophilic methane, and microaerophilic methanol
    • Each experimental group was done in triplicate
  • Recorded the OD at hour 0 and shook overnight at 30 ºC

Wednesday

  • Continued taking ODs for the growth assay
  • Skyped with UT Austin’s iGEM team about their project and collaborating
    • This time with 12 tubes: oxic methane, oxic methanol, anoxic methane, anoxic methanol
    • The anoxic experimental groups were done in the anaerobic chamber for the first time
  • Recorded the OD at hour 0 and shook overnight at 30 ºC
  • Checked plates for transformation and set-up colony PCR from the colonies that grew

Thursday

  • Continued taking optical density for growth assay
  • Checked colony PCR with a gel
    • Did not have a band at the right size
  • Redid transformation with the same Gibson Assemblies but used commercial competent E. Coli cells
    • Did the transformation via electroporation
    • Plated Lb + Kan plates and grew overnight

Friday

  • None of the Gibson colonies grew
  • Did Gibson Attempt 2 and 3 with a 3:1 ratio and 1:1 ratio of insert to backbone
  • The following tables show the 3:1 ratio additions:

Gibson 1 (Construct A) :

Fragment Amount (uL)
pmoA- RBS-nir 1 uL
RBS-DAMO_2434 1 uL
pAWP78 backbone 1.3 uL

Gibson 2 (Construct B) :

Fragment Amount (uL)
pmoA- RBS-nir 1 uL
RBS-DAMO_2437 1 uL
pAWP78 backbone 1.3 uL

Gibson 3 (Construct 3) :

Fragment Amount (uL)
pmoA-RBS-nir 1 uL
RBS-DAMO_2434 1 uL
RBS-DAMO_2437 1 uL
pAWP78 backbone 1.1 uL
Week 5

06/16 - 06/22

Monday

  • Transformation of 3:1 and 1:1 assembly ratios via electroporation
    • Only used 40 uL of competent cells per transformation
    • Added 60 uL of dd h 2O
    • Shake at 600 rpm at 37 ºC for one hour
    • Plated on Lb + Kan plates and put to grow overnight at 37 ºC

Tuesday

  • Colonies grew on plates from construct B and C, but not A
  • Did colony PCR using 4 colonies from each plate
  • Ran a gel:
  • Bands were not what we were expecting and ladder wasn't separating well so we run another

Wednesday

  • Did another colony PCR from colonies that had grown on our negative control to use for reference
  • Realized we accidentally froze our colonies we had separated before we did PCR
    • Took out of freezer to shake to see if they could be recovered
    • They did not grow so we will redo transformation with the remaining Gibson assemblies tomorrow
  • Ran a gel with negative control
  • We did get bands that we wanted but since we no longer have the colonies we need to do another transformation

Thursday

  • Did transformation via electroporation
    • Did 7 transformations, 3 3:1 ratios, 3 1:1 ratios and a negative control
  • Will grow plates overnight and at 37 ºC and check for colonies in the morning

Friday

  • Made P media, autoclaved tubes and stoppers and put into glovebox for future growth experiments
  • Colony PCR on colonies on each plate, ran a gel, we believe we have the plasmid inserted into the bacteria for the B construct
Week 6

06/23 - 06/29

Monday

  • Determined that the B3 construct is likely to have the plasmid based on our gel
  • Streaked out culture on LB + Kan plate from our spotted plate of B32 and B33
  • Put plate at 37 degrees to grow isolated colonies overnight so we can purify our plasmid tomorrow!
  • Made 100 mL of P media with 1.5 g agar to pour plates
  • Did Gibson Assembly of 3:1 ratios of Construct A and C
  • Inoculated two tubes of Kan + LB with the plasmid B33 and B32, set to shake in the 30 C room overnight

Tuesday

  • The inoculated tubes that were shaking had many particles in them but we put them back to shake longer to see if more growth would occur
  • The struck out plate from yesterday did not appear to have pure colonies so we struck out another plate in an attempt to get pure independent colonies
  • Put the plate at 37 ºC overnight
  • Transformation of A and C constructs in Mock 1 cells and then plated on Lb + Kan
  • Streaked P media agar plate with 20 Z

Wednesday

  • Took colonies from plates from yesterday (B construct) and put them to shake at 30 ºC in 3 mL of LB + Kan
  • Began setting up growth assay
  • Ran Plasmid Purification with the “Failed” inoculated tubes, used the nanodrop to see concentration of plasmid
    • These were the tubes that had a lot of unexpected particles that were growing
  • Inoculated tubes in anoxic chamber for another growth assay, adding carbon sources methanol and methane and took ODs

Thursday

  • Took ODs
  • Struck out a new plate from original B3 plate with four colonies to see if we can get more independent colonies to try growth again with a newly prepared media

Friday

  • Took ODs
  • Transformed A and C into lab-made competent cells and plated
  • Put new B construct colonies to grow with newly made media

July

Week 7

06/30 - 07/06

Monday

  • Realized the dilutions we did for Gibson were not carried out correctly, so we redid Gibson cloning for constructs A,B,C
  • The Gibson was done in a 1:1 ratio and 2:1 ratio of insert to backbone (but calculations done properly this time)

Tuesday

  • Transformation into D-alpha cells of assemblies from previous day
  • Plated on LB + Kan plates and incubating at 37 ºC overnight

Wednesday

  • Continued to let the plates with the transformed constructs grow and then put them in the fridge over the long weekend
Week 8

07/07 - 07/13

Monday

  • Since no colonies have been growing after our many attempts of Gibson we have been troubleshooting
  • Reviewing issues related to our backbone

Tuesday

  • DPN1 digest of “clean BB” to be sure all circular plasmid has been digested
  • Repeated Gibson Assembly with Constructs A and B with a higher concentration of DNA

Wednesday

  • Diluted Primers
  • PCR optimization of inserts to get more concentrated DNA
  • Repeated PCR optimization of inserts 24 and 27 using 9% DMSO because they did not get strong bands
    • Ran a gel to see size of inserts
  • Transformation of Gibson from previous day

Thursday

  • Ran a gel to determine if 9% DMSO PCR reaction worked for insert 24 and 27
  • Ran a gel for gel extraction with all inserts
  • Completed gel extraction/purification of inserts 21, 22, 23, and 26
  • Completed PCR of 24, 25 and 27 because we weren’t able to extract from the gel
  • Completed cPCR of colonies from transformation from plate B
  • Spotted colony from B construct on a plate to grow overnight

Friday

  • Gel for PCR optimization of inserts 24, 25, 27
  • Purification of extracted gel to send for sequencing
  • Gibson Assembly in a 3:1 ratio and transformation for Construct A
Week 9

07/14 - 07/20

Monday

  • PCR of inserts 24 25 and 27 -- unsuccessful gel (may be trying with MgCl2 gradient)
  • Streaked out a new plate of B construct to get isolated colonies
  • Prepared concentrations for future SLiCE Procedure

Tuesday

  • Ran colony PCR gel of construct B
  • PCR of 24,25 using concentrations of MgCl
  • Performed SLiCE on A and B constructs
  • Transformed and plated A and B constructs
  • Inoculated two tubes of LB + Kan media with the B construct

Wednesday

  • Made more LB + Kan plates
  • More transformations of the SLiCE mixtures doing both electroporation and Heat Shock
  • Ran a gel of the PCR products of 24 25 for gel extraction and purification
  • Purified constructs 24 25 and took concentrations using the nanodrop
  • Reinoculated LB + Kan solutions with independent colonies from the B Construct Plate

Thursday

  • Completed a PCR for constructs 24 25 27 using a MgCl2 gradient
  • Ran a gel to check sizes of 24 25 27 constructs and then ran another gel to complete gel extraction

Friday

  • Completed gel extraction and purification of 21 22 23 27
  • Did PCR of 24 using MgCl2 and ran a gel to see the size of the band
  • Ran a gel of the B Construct from plasmid extraction
  • Purified Plasmid from LB + Kan shaking the construct B twice and used the nanodrop to get the concentration
Week 10

07/21 - 07/27

Monday

  • Gel extraction of insert 24 and purification
  • 260/230 numbers look off so redoing PCR of inserts 21 22 23 and holding off on sequencing
  • Perform Gibson assembly for constructs A,B
  • Transform Gibson Assemblies
  • Run a gel with Gibson assemblies to determine size and to troubleshoot
  • Transform construct B into s17 using 3 uL of purified plasmid (14 ng/uL)

Tuesday

  • Ran a gel for PCR of 21 22 23
  • Put LB + Kan and tubes in the anoxic chamber for growing construct B plasmid under anoxic conditions

Wednesday

  • Ran a gel for PCR of 21 22 23
  • Ran a gel for gel exactration of 21 22
  • Learned how to operate the Cell Disruption mechanism (French Press)

August

Week 11

08/04 - 08/10

No activity

Week 12

08/11 - 08/17

No activity

Week 13

08/18 - 08/24

No activity

Week 14

08/25 - 08/31

Tuesday

  • PCR of all inserts and gel extraction

September

Week 15

09/01 - 09/07

No activity

Week 16

09/08 - 09/14

Sunday

  • PCR of backbone, failed, continued into next week

      • Thursday

      • PCR/gel extraction of pAWP78
Week 17

09/15 - 09/21

Tuesday

  • Gibson assembly and transformation of construct B, failed

Thursday

  • Transformation of construct B into DH5alpha, success

Friday

  • Colony PCR of contruct B colonies
Week 18

09/22 - 09/28

Tuesday

  • Plasmid purification from cultures
  • PCR of DNA from plasmid purification

Wednesday

  • PCR (colony PCR) of purified plasmid
  • Sequencing set up

October

Week 19

09/29 - 10/05

Monday

  • Made 20Z competent cells
  • Transformed purified plasmid into 20Z
  • Sent out plasmid for sequencing
  • Setting up for growth experiment