Difference between revisions of "Team:UMaryland/Notebook"

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<div id='contentbox'>
 
<div id='contentbox'>
 
<a name="Week1">
 
<a name="Week1">
−
<p style="font-size:32px;text-align:center;font-family:Verdana, Geneva, sans-serif;">
+
<p style="font-size:32px;text-align:left;font-family:Verdana, Geneva, sans-serif;">
 
<b>Week 1</b></a>
 
<b>Week 1</b></a>
−
<p style="font-size:24px;text-align:center;">
+
<p style="font-size:24px;text-align:left;">
 
Miraculin:
 
Miraculin:
 +
<br>
 
ligated the pBAD promoter in PSB1C3 with the miraculin gene in PSB1C3 using 3A assembly
 
ligated the pBAD promoter in PSB1C3 with the miraculin gene in PSB1C3 using 3A assembly
 +
<br>
 
plate had colonies
 
plate had colonies
 
<br>
 
<br>
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<div id='contentbox'>
 
<div id='contentbox'>
 
<a name="Week2">
 
<a name="Week2">
−
<p style="font-size:32px;text-align:center;font-family:Verdana, Geneva, sans-serif;">
+
<p style="font-size:32px;text-align:left;font-family:Verdana, Geneva, sans-serif;">
 
<b>Week 2</b></a>
 
<b>Week 2</b></a>
−
<p style="font-size:24px;text-align:center;">
+
<p style="font-size:24px;text-align:left;">
 
Miraculin:
 
Miraculin:
−
pBAD +miraculin construct was moved back into psb1C3 backbone and sent for sequencing
+
<br>
 +
The pBAD +miraculin construct was moved back into psb1C3 backbone and sent for sequencing
 +
<br>
 
Designing gBlocks:
 
Designing gBlocks:
 +
<br>
 
Designed gblocks for epsilon-cyclase (from arabidopsis), pyocin and Hok/Sok  
 
Designed gblocks for epsilon-cyclase (from arabidopsis), pyocin and Hok/Sok  
 
<br>
 
<br>
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<div id='contentbox'>
 
<div id='contentbox'>
 
<a name="Week 3">
 
<a name="Week 3">
−
<p style="font-size:32px;text-align:center;font-family:Verdana, Geneva, sans-serif;">
+
<p style="font-size:32px;text-align:left;font-family:Verdana, Geneva, sans-serif;">
 
<b>Week3</b></a>
 
<b>Week3</b></a>
−
<p style="font-size:24px;text-align:center;">
+
<p style="font-size:24px;text-align:left;">
 
Miraculin:
 
Miraculin:
 +
<br>
 
sequence confirmed through sequencing  
 
sequence confirmed through sequencing  
 +
<br>
 
failed to extract using French press, FPLC and SDS-Page  
 
failed to extract using French press, FPLC and SDS-Page  
 +
<br>
 
could be due to high arabinose induction (OD of 1)
 
could be due to high arabinose induction (OD of 1)
  
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<div id='contentbox'>
 
<div id='contentbox'>
 
<a name="Week4">
 
<a name="Week4">
−
<p style="font-size:32px;text-align:center;font-family:Verdana, Geneva, sans-serif;">
+
<p style="font-size:32px;text-align:left;font-family:Verdana, Geneva, sans-serif;">
 
<b>Week 4</b></a>
 
<b>Week 4</b></a>
−
<p style="font-size:24px;text-align:center;">
+
<p style="font-size:24px;text-align:left;">
 
Miraculin:  
 
Miraculin:  
 +
<br>
 
induced 3 test cultures with  0%, 0.05%, 0.1%, 0.2% arabinose with no significant results
 
induced 3 test cultures with  0%, 0.05%, 0.1%, 0.2% arabinose with no significant results
 +
<br>
 
will perform procedure again using bl21 instead of Dh5a
 
will perform procedure again using bl21 instead of Dh5a
  
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<div id='contentbox'>
 
<div id='contentbox'>
 
<a name="Week5">
 
<a name="Week5">
−
<p style="font-size:32px;text-align:center;font-family:Verdana, Geneva, sans-serif;">
+
<p style="font-size:32px;text-align:left;font-family:Verdana, Geneva, sans-serif;">
 
<b>Week 5</b></a>
 
<b>Week 5</b></a>
−
<p style="font-size:24px;text-align:center;">
+
<p style="font-size:24px;text-align:left;">
−
Miraculin
+
Miraculin:
 +
<br>
 
transformation in BL21 strain was successful
 
transformation in BL21 strain was successful
 +
<br>
 
SDS page showed a band at roughly 25 kDa
 
SDS page showed a band at roughly 25 kDa
 +
<br>
 
Hok/Sok
 
Hok/Sok
 +
<br>
 
inserted Hok/Sok gblock into PSB1C3 backbone using Gibson assembly
 
inserted Hok/Sok gblock into PSB1C3 backbone using Gibson assembly
 +
<br>
 
construct sent for sequencing
 
construct sent for sequencing
 +
<br>
 
Interlab Study
 
Interlab Study
 +
<br>
 
transformed parts into dh5a: 3 Anderson promoters, GFP, and RFP
 
transformed parts into dh5a: 3 Anderson promoters, GFP, and RFP
  
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<div id='contentbox'>
 
<div id='contentbox'>
 
<a name="Week6">
 
<a name="Week6">
−
<p style="font-size:32px;text-align:center;font-family:Verdana, Geneva, sans-serif;">
+
<p style="font-size:32px;text-align:left;font-family:Verdana, Geneva, sans-serif;">
 
<b>Week 6</b></a>
 
<b>Week 6</b></a>
−
<p style="font-size:24px;text-align:center;">
+
<p style="font-size:24px;text-align:left;">
−
Miraculin:  
+
Hok/Sok:
−
induced 3 test cultures with 0%, 0.05%, 0.1%, 0.2% arabinose with no significant results
+
<br>
−
will perform procedure again using bl21 instead of Dh5a
+
Transformed unstable GFP and RFP with PBAD into dh5a
 +
<br>
 +
Transformed unstable RFP and inducible lac promoter (k1399011) and const_promoter+RBS (K081005) into dh5a
 +
<br>
 +
ligated const_promoter + RBS + RFP
 +
<br>
 +
Interlab:
 +
<br>
 +
performed a 3A assembly of each promoter + GFP in PSB1K3 (GFP in PSB1A3 and promoters in PSB1C3)
  
  
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<div id='contentbox'>
 
<div id='contentbox'>
 
<a name="Week7">
 
<a name="Week7">
−
<p style="font-size:32px;text-align:center;font-family:Verdana, Geneva, sans-serif;">
+
<p style="font-size:32px;text-align:left;font-family:Verdana, Geneva, sans-serif;">
 
<b>Week 7</b></a>
 
<b>Week 7</b></a>
−
<p style="font-size:24px;text-align:center;">
+
<p style="font-size:24px;text-align:left;">
 +
Hok/Sok:
 +
<br>
 +
construct created through 3A assembly of quick degrading RFP and constitutive promoter +RBS was proven incorrect using sequencing
 +
<br>
 +
Sequencing showed a 3A assembly of quick degrading RFP and a "leaky" lactose promoter
 +
<br>
 +
Re- ran 3A assembly but the transformation failed
 +
<br>
 +
could be due to contaminated SOC media
  
 
<br>
 
<br>
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<div id='contentbox'>
 
<div id='contentbox'>
 
<a name="Week8">
 
<a name="Week8">
−
<p style="font-size:32px;text-align:center;font-family:Verdana, Geneva, sans-serif;">
+
<p style="font-size:32px;text-align:left;font-family:Verdana, Geneva, sans-serif;">
 
<b>Week 8</b></a>
 
<b>Week 8</b></a>
−
<p style="font-size:24px;text-align:center;">
+
<p style="font-size:24px;text-align:left;">
  
 +
Hok/Sok
 +
<br>
 +
const_promoter + RBS + RFP were replated from last week but produced no colonies
 +
<br>
 +
re-transformed original and new 3A assembly which produced the correct sequence
 +
<br>
 +
ligated const_promoter:QD-RFP in PSB1C3
 +
<br>
 +
performed a 3A assembly of Hok/Sok plasmid with QD-RFP downstream in PSB1K3
 +
<br>
 +
site directed mutagenesis of quick degrading GFP (Bba_K750000)  failed
 +
<br>
 +
there was no change from the original sequence
 +
<br>
 +
Interlab study
 +
<br>
 +
3A assembly attempts for the promoters and GFP failed
 +
<br>
 +
Gibson Assemblies produced a vast amount of colonies
 +
<br>
 +
questionable success because the amount of colonies may indicate false positives or contamination
 +
<br>
 +
Lutein
 +
<br>
 +
ordered pAC-LYC which encodes 3 enzymes from E. Herbicola to produce basal levels of lycopene
 +
<br>
 +
PCR
 +
<br>
 +
code made and proven to properly cycle machine
  
 
<br>
 
<br>
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<div id='contentbox'>
 
<div id='contentbox'>
 
<a name="Week9">
 
<a name="Week9">
−
<p style="font-size:32px;text-align:center;font-family:Verdana, Geneva, sans-serif;">
+
<p style="font-size:32px;text-align:left;font-family:Verdana, Geneva, sans-serif;">
 
<b>Week 9</b></a>
 
<b>Week 9</b></a>
−
<p style="font-size:24px;text-align:center;">
+
<p style="font-size:24px;text-align:left;">
 +
Hok/Sok
 +
<br>
 +
previous 3A assembly from last week showed no colony growth on kanamycin plate
 +
<br>
 +
transformed RFP into C3 backbone last week
 +
<br>
 +
colonies were produced that were not distinctively red but with the correct sequence
 +
<br>
 +
3A assembly with the RFP + Hok/Sok failed
 +
<br>
 +
Created primers for Gibson assembly to create Hok/Sok +const_promoter::RBS::unstable RFP
 +
<br>
 +
Interlab Study
 +
<br>
 +
replated last week’s constructs with GFP at lower concentration selected for chloramphenicol which was successful
 +
<br>
 +
K08  and K13 constructs grew colonies
 +
<br>
 +
Miniprepped constructs and only promoter K08+GFP had the correct sequence
 +
<br>
 +
PCR
 +
<br>
 +
received high rated peltier units that did not break
 +
<br>
 +
took ~31 minutes to complete 1 cycle but insulated heating dropped the time to 16 minutes
 +
<br>
 +
Took apart a hair dryer for heating element
 +
<br>
 +
heating went to >95 in ~2 seconds, cooled to 50 in ~8 seconds
 +
<br>
 +
The heating element is not very precise so it resulted in a lot of overshoot in temperature
  
 
<br>
 
<br>
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<div id='contentbox'>
 
<div id='contentbox'>
 
<a name="Week10">
 
<a name="Week10">
−
<p style="font-size:32px;text-align:center;font-family:Verdana, Geneva, sans-serif;">
+
<p style="font-size:32px;text-align:left;font-family:Verdana, Geneva, sans-serif;">
 
<b>Week 10</b></a>
 
<b>Week 10</b></a>
−
<p style="font-size:24px;text-align:center;">
+
<p style="font-size:24px;text-align:left;">
 +
Hok-Sok
 +
<br>
 +
created construct with RFP and hok/sok
 +
<br>
 +
Pcr of the construct failed so the construct will be sequenced to check
 +
<br>
 +
PCR /Gibson Check
 +
<br>
 +
performed a series of check in order to ensure that all the reagents are active
 +
<br>
 +
everything seems to be working
 +
<br>
 +
PCR machine
 +
<br>
 +
cycle data was taken and it exhibits consistency
  
 
<br>
 
<br>
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<div id='contentbox'>
 
<div id='contentbox'>
 
<a name="Week11">
 
<a name="Week11">
−
<p style="font-size:32px;text-align:center;font-family:Verdana, Geneva, sans-serif;">
+
<p style="font-size:32px;text-align:left;font-family:Verdana, Geneva, sans-serif;">
 
<b>Week 11</b></a>
 
<b>Week 11</b></a>
−
<p style="font-size:24px;text-align:center;">
+
<p style="font-size:24px;text-align:left;">
 +
Hok/Sok
 +
<br>
 +
3A assembly of H/S + RFP failed
 +
<br>
 +
ran a PCR of PSB1C3+H/S+RFP using 3 different rxn buffers
 +
<br>
 +
HF rxn buffer
 +
<br>
 +
GC rxn buffer
 +
<br>
 +
GC rxn buffer w/ DMSO which yielded product
 +
<br>
 +
Ran a PCR of unstable RFP using the  3 rxn buffers above
 +
<br>
 +
Interlab Study
 +
<br>
 +
used Phusion instead of Q5 for PCR
 +
<br>
 +
ran PCR for GFP 1, GFP 3, and IL1 which produced the correct sized products
 +
<br>
 +
Gibson Assembly of GFP + IL1 produced colonies
 +
<br>
 +
PSB1C3 - IL3 did not have bands in gel of appropriate size
 +
<br>
 +
Lutein
 +
<br>
 +
RE digests of pLAC-RFP in PSB1C3
 +
<br>
 +
ran a Gibson Assembly of E-cyclase/C3 and E-hydroxylase/C3 which produced colonies
 +
<br>
 +
PCR Machine
 +
<br>
 +
rebuilt top of hair dryer housing w/ soda can
 +
<br>
 +
observed random temp spikes occurring during each run
 +
<br>
 +
caused by hardware issue with relays
 +
<br>
 +
purchased new relays to test this week
  
  
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<div id='contentbox'>
 
<div id='contentbox'>
 
<a name="Week12">
 
<a name="Week12">
−
<p style="font-size:32px;text-align:center;font-family:Verdana, Geneva, sans-serif;">
+
<p style="font-size:32px;text-align:left;font-family:Verdana, Geneva, sans-serif;">
 
<b>Week 12</b></a>
 
<b>Week 12</b></a>
−
<p style="font-size:24px;text-align:center;">
+
<p style="font-size:24px;text-align:left;">
 +
Hok/Sok:
 +
<br>
 +
Ran a Gibson Assembly of Hok/Sok + RFP
 +
<br>
 +
Interlab
 +
<br>
 +
worked on finishing the last construct
 +
<br>
 +
K13 sequenced correctly and the pcr looks good
 +
<br>
 +
contacted W&M on possible collaboration for the last construct
 +
<br>
 +
Lutein
 +
<br>
 +
ordered primers for Gibson Assembly
 +
<br>
 +
PCR
 +
<br>
 +
replaced old relays with higher powered relays
 +
<br>
 +
reduced mass by over 50% of the peltier machine
 +
<br>
 +
resulted in speedier temperature changes
  
 
<br>
 
<br>
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<div id='contentbox'>
 
<div id='contentbox'>
 
<a name="Week13">
 
<a name="Week13">
−
<p style="font-size:32px;text-align:center;font-family:Verdana, Geneva, sans-serif;">
+
<p style="font-size:32px;text-align:left;font-family:Verdana, Geneva, sans-serif;">
 
<b>Week 13</b></a>
 
<b>Week 13</b></a>
−
<p style="font-size:24px;text-align:center;">
+
<p style="font-size:24px;text-align:left;">
−
Miraculin:
+
Hok/Sok
 +
<br>
 +
sequence of hok/sok construct created last week was incorrect
 +
<br>
 +
may put a unique RE site between hok/sok and rfp when retrying 3A assembly, RE cloning and Gibson
 +
<br>
 +
Interlab
 +
<br>
 +
Gibsons of IL3 (K13 + GFP) have all failed
 +
<br>
 +
3 trials of IL2 , IL1,+ control (const_promoter with GFP), - control were tested using the plate reader
 +
<br>
 +
IL2 was a little low in fluorescence, similar to the - control
 +
<br>
 +
PCR
 +
<br>
 +
Rewired hair dryer and changed relays to handle higher wattage
 +
<br>
 +
attempted PCR cycle failed
 +
<br>
 +
assumed temp sensor needed to be immersed in mineral oil to properly report temp
 +
<br>
 +
denaturation temp may have been too low
 +
  
  
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<div id='contentbox'>
 
<div id='contentbox'>
 
<a name="Week14">
 
<a name="Week14">
−
<p style="font-size:32px;text-align:center;font-family:Verdana, Geneva, sans-serif;">
+
<p style="font-size:32px;text-align:left;font-family:Verdana, Geneva, sans-serif;">
 
<b>Week 14</b></a>
 
<b>Week 14</b></a>
−
<p style="font-size:24px;text-align:center;">
+
<p style="font-size:24px;text-align:left;">
−
Miraculin:  
+
Hok/Sok
−
 
+
<br>
 +
Began testing for RFP fluorescence for Hok/Sok effectiveness as a plasmid maintenance system
 +
<br>
 +
Group A: RFP coding region; constitutive promoter ; chloramphenicol in liquid culture
 +
<br>
 +
Group B: RFP coding region; constitutive promoter ; no chloramphenicol in liquid culture
 +
<br>
 +
Group C: RFP coding region; no promoter ; chloramphenicol in liquid culture
 +
<br>
 +
Group D: H/S-RFP coding region; constitutive promoter ; chloramphenicol in liquid culture
 +
<br>
 +
Group E: H/S-RFP coding region; constitutive promoter ; nochloramphenicol in liquid culture
 +
<br>
 +
Group F: H/S-RFP coding region; constitutive promoter ; chloramphenicol in liquid culture
 +
<br>
 +
Interlab:
 +
<br>
 +
Obtained the last construct from W&M
 +
<br>
 +
Tested the last construct using the plate reader
 +
<br>
 +
Turned in the IL study
  
  
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<div id='contentbox'>
 
<div id='contentbox'>
 
<a name="Week15">
 
<a name="Week15">
−
<p style="font-size:32px;text-align:center;font-family:Verdana, Geneva, sans-serif;">
+
<p style="font-size:32px;text-align:left;font-family:Verdana, Geneva, sans-serif;">
 
<b>Week 15</b></a>
 
<b>Week 15</b></a>
−
<p style="font-size:24px;text-align:center;">
+
<p style="font-size:24px;text-align:left;">
−
Miraculin:
+
  
 +
Hok/Sok
 +
<br>
 +
Tested generations alpha beta and gamma in BL21 cells
 +
<br>
 +
alpha and beta contained groups A-E
 +
<br>
 +
gamma had only group F
  
  
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<div id='contentbox'>
 
<div id='contentbox'>
 
<a name="Week16">
 
<a name="Week16">
−
<p style="font-size:32px;text-align:center;font-family:Verdana, Geneva, sans-serif;">
+
<p style="font-size:32px;text-align:left;font-family:Verdana, Geneva, sans-serif;">
 
<b>Week 16</b></a>
 
<b>Week 16</b></a>
−
<p style="font-size:24px;text-align:center;">
+
<p style="font-size:24px;text-align:left;">
−
Miraculin:
+
 
 +
Hok/Sok
 +
<br>
 +
Tested generation delta using DH5alpha
 +
<br>
 +
contained groups A-E
 +
 
  
  

Revision as of 08:51, 18 September 2015