Team:Stanford-Brown/SB16 Notebooks Chromoproteins


Stanford-Brown 2016

Chromoproteins · Benchling

Chromoproteins

Made with Benchling
Project: iGEM 2016
Authors: Cynthia Hale-Phillips, Theresa Sievert, Taylor Pullinger
Dates: 2016-06-06 to 2016-10-18
Monday, 6/6/16
Students arrive! Orientation to Rothschild Astrobiology/Synthetic Biology Lab at NASA Ames Research Center
Plan to continue research on chromoproteins to catalogue the denaturation temperature of each protein
If we do so, perhaps we can employ it in a biological thermometer
Tuesday, 6/7/16
E. coli expressing producing different chromoproteins were placed into 15 mL tubes with LB and incubated at 37 °C for 24 hours.
A
B
1
ChromoproteinObservations
2
RFPClear red color, fluorescent
3
amil GFP (yellow)Yellow color, fluorescent
4
amil CP (indigo)Purple blue color, not fluorescent
5
meff (blue)No significant color production
iGEM Chromoprotein Color and Fluorescent Observations
The RFP was examined for whether it was heat or light inactivated. Our rudimentary setup to analyze this was to extract a portion of the cells and smear them onto a clear plate. This was then heated to 50 °C, at which point the cells dried and also lost their color. In addition, it was no longer fluorescent.
The cells were allowed to sit for a short period of time until the temperature had dropped, upon which the RFP appeared to slowly regain pink color and became fluorescent again.
These preliminary results into the nature of RFP suggest that it is heat-inactivated, meaning that there is definitely potential for application in a bio-thermometer.
Wednesday, 6/8/16
Pellet the liquid cultures started yesterday by centrifuging
Transferred portions of each pellet into PCR tubes, then heated them at different temperatures in thermal cycler to see where the color loss for each chromoprotein was
RFP denatures at 90˚C
Indigo denatures at 90˚C
Thursday, 6/9/16
The heated RFP did not renature overnight, remained discolored
Tested fluorescence for RFP and GFP
Performed Gibson assembly of the 4 chromoproteins for iGEM (AmiGFP Yellow, RFP Red, meffblue - Blue, AmilCP - Indigo) with primers ordered last week
Then transformed these into chemically competent E. Coli and plated these Gibson assemblies onto LB+chlor plates
Friday, 6/10/16
Gibson assembly iGEM chromoproteins: No growth on any of the LB+chlor plates
Due to a mistake in the sequence when we ordered the primers
New, correct primers have been ordered
Monday, 6/13/16
Used new primers to PCR our 4 iGEM chromoprotein and his-tag to get them ready for a Gibson assembly tomorrow
Tuesday, 6/14/16
Performed Gibson assembly on the 4 iGEM chromoproteins and transformed and plated and placed into the 37 °C incubator overnight and will hopefully see growth on plates tomorrow
Wednesday, 6/15/16
No growth on any of the plates with the new Gibson assembly
Perhaps the chromoprotein sequence is incorrect
Thursday, 6/16/16
Cell competency test done on 3 different DNA concentrations (0.5pg/ul, 10pg/ul, 50pg/ul) with 2 different cell types (T7, 5-alpha)
Protocol given for the competency test wasn't compatible with T7, but we got colonies on all of our 5-alpha plates (Charlie, Taylor)
Friday, 6/17/16
Started new Gibson assembly of amilCP and the flag lumio-His tags
Then trasnformed and plated this Gibson assembly
Monday, 6/20
Checked last week's Gibson assembly on chromoprotein and flag lumio-His tags. No colonies across all 3 tested plates.
Tuesday, 6/21
Gibson redo #3
Diluted tag from 577 ng/ul to 150 ng/ul to get 2.9 pmol/ul
1 ul of dilution and 1ul of vector added together
2 types of incubation: 50 ˚C for 15 min and stepped down from 52 ˚C (0.5 ˚C/5 min) until temp reached 48 ˚C
Stepped down temperature labeled as #2
Transformed with NEB 5-alpha and plated with 1:4 and 1:40 dilution
Gibson: discovered we initially didn't ligate the strands properly with buffer, Cynthia is doing a redo
Wednesday, 6/22
Gibson assembly re done today and strands properly ligates with buffer
Thursday, 6/23
Gibson worked!!!!!!!!!! Colony growth seen on plates
Monday, 6/27
We obtained chromoproteins from DNA 2.0 in the paintbox set
Here is a link to more information on the different chromoproteins contained in the paintbox: https://www.dna20.com/eCommerce/catalog/datasheet/348.
For our purposes we used the kanamycin resistance.
We plan on inserting these chromoproteins into a psB1C3 backbone to standardize to iGEM
A
B
C
1
Chromoprotein NameShorthand GivenDNA 2.0 name
2
Blitzen BlueBBCBP-33-444
3
Dreidel TealDTCBP-34-444
4
Virginia VioletVVCBP-35-444
5
Vixen PurpleVPCBP-36-444
6
Prancer PurplePPCBP-37-444
7
Tinsel PurpleTPCBP-38-444
8
Maccabee PurpleMPCBP-39-444
9
Donner MagentaDMCBP-40-444
10
Cupid PinkCPCBP-41-444
11
Seraphina PinkSPCBP-44-444
12
Scrooge OrangeSOCBP-45-444
13
Leor OrangeLOCBP-46-444
Table2
Here are the 12 DNA2.0 Chromoproteins we will be working with and the abbreviations we will be using throughout the summer for each of them
We created primers for all of these chromoproteins for the vector (all the same), its promoter, the specific chromoprotein, and the cellulose binding domains that will be included into the chromoproteins.
Because the chromoproteins shared many cellulose binding domain primers they were named to show which one was shared and could be used multiple times.
A
B
C
1
ShorthandSequenceUsed For
2
CBD1GTCATCACAAAACCGGCGGTBB
3
CBD2TCATAACACCGGCGGTCCGDT
4
CBD3TCATCACACCGGCGGTCCGVV, DM, LO
5
CBD4AGCGACGACCGGCGGTCCGVP, PP, TP, CP
6
CBD5AGCGACCACCGGCGGTCCGSP, MP
7
CBD6AGAAACGACCGGCGGTCCGSO
Table1
Table3: shows the shorthand used for the forward cellulose binding domain primers
Wednesday, 6/29
Started 5 ml liquid cultures from the iGEM biobrick chromoproteins to see if we could induce the color we expected
Did PCR for the Gibson Assembly tomorrow on the linearized pSB1C3, cellulose binding domain (CBD), flag lumio His-tag (His-tag) and the DNA2.0 chromoproteins. The primers for these reactions are in the chromoprotein excel spreadsheet
Thursday, 6/30
Ran a gel with the 12 chromoproteins that had been PCRed and CBD1-6
Performed gel extraction on all 18 samples
Chromoproteins MP and LO did not show bands of DNA in the right place on the gel for this reason we decided to redo the primers for them
Tuesday, 7/5
We performed a Gibson assembly on the DNA 2.0 chromoproteins to add a CBD and His-tag all together
A
B
C
D
E
F
G
H
I
J
1
Gibson Assembly Amounts Added
2