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Q2 – Week 6 – 12/20 – 12/23

                                                                                                                             Jump to:  Tuesday,   Wednesday,  Thursday,  Friday                                                                                                                                   ___________________________________________________________

12/20 – Monday –  A Day

Main focus –   
                                                                                                                                           
a)  To identify a missense, silent, and nonsense (point) mutations.
                                                 
b)  To identify frameshift mutations (insertion & deletions.
c)  To begin the pGlo lab by streaking our plates.

1: We will review the Linked Genes 2 form and the Gene to Protein Form.

2. Mutations – completed the Mutation worksheet together

Mutations – 2016.pdf                                                                                                                                                                View Download

Substitutional Mutations:

Missense – 
Silent – 
Nonsense – 
 
Reading Frame Mutations:
Insertions – 
Deletions – 

3: Gene Regulation Lecture – We did not do this in class today.

                 a) Trp operon

                  b) Lac Operon

Gene Regulation Diagrams new 1819.pdf
View Download

4: Streak plates for pGlo Lab   –  We did streak our plates.      

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12/20 – Monday’s Homework: –

1.  Please view the Trp and Lac Operon Lecture.

2. Complete the form based on the lecture and or the textbook hints given.  The form will NOT be on auto-reply. Make your 1 submission your best!

1: Trp and Lac Operon Lecture:

 
2: Regulation of  Gene Expression

End of Monday..

 

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12/21 – Tuesday –  B Day – 

Main focus –                                                                                                                                                          
a)  To Review the concepts of translation and transcription and connect it to the gene regulation of the Trp and Lac operon.    
                                                                                                                                                                                                             b)  To Review a repressable and inducible operon.                                                                                                                                                                                                                                                                      1.  Trp operon / Lac operon–                                                                                                                                                                                                                                                                                                                                                   2.  pGlo Lab –   culture the four test plates.                                                                                                                                                                                                                                                                                           
1: pGlo Transformation Lab- 
 
    a) obtain 2 micro test tubes, one labeled +pGlo and the other -pGlo
         Little p means plasmid – small circular DNA of E. Coli.
 
    b) Obtain your starter plates that you streaked yesterday that has been incubating for 24 hours at 37 degrees   
         Celsius.
 
    c) Use a sterile loop and pick up a single colony and disperse into each tube, +pGLO and  -pGlo.
 
     d) I will add 10 micro liters of the pGlo plasmid (engineered sequence) to your +pGLO micro test tube.
 
    e) Incubate the tubes on ice for 10 minutes.
 
    f) label your lids.
 
    g) heat shock, add broth, pippete and streak in the appropriate plates.
 
    f) Stack up the plates, tape and label, then incubate for 24 hours.

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Trp Operon Notes: 

 The Trp Operon is segment on the bacterial DNA includes a single promoter for a group of genes.  These genes represent the 5 enzymes in a single metabolic pathway that will produce tryptophan.  This is an anabolic operon as it builds or synthesizes the amino acid tryptophan from other resources. It is an repressible operon that is on the “on” position until a repressor protein produced from an upstream regulatory gene that will turn the operon “off” when there is enough tryptophan present in the cell.

The switch of the operon is the operator position in the promoter region that the regulatory protein (repressor) can bind to and will prevent RNA polymerase from binding to promoter and turn the operon “off”.   

It is a negative control operon.

                                                                   

                                                                                                                                                                  Metabolic pathway in the synthesis of tryptophan:                                                                                     
https://www.researchgate.net/publication/225551231_Tryptophan_Biosynthesis_in_Stramenopiles_Eukaryotic_Winners_in_the_Diatom_Complex_ChloroplastER –                                                                                                                                                                                                                 

Trp Operon Animation:

pGlo Lab Instructions:

  _____________________

12/21 – Tuesday’s Homework: –

1. Please make 2 more submissions to the Gene Regulation Form.  I resent you the new grades AFTER class due to an error in my original key. Thanks Zeke! I DEEPLY appreciate it! 

Please refresh this page to see the new Changes..                                                                     

2:  Please read the pGlO lab (transformation lab) pages 1 – 11 in your lab packet and complete pGlo form below:

 
Transformation Lab instructions – student manual.pdf
View Download
There will only be one submission!
                                                                                                                                                                                                            
 
2: pGlo Lab form

End  of Tuesday…

 

 

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12/21 – Wednesday –  A Day 

Main focus –                                                                                                                                                          
a) To complete the pGlo Lab by observing our test plates.
b)  To review Trp Operon and Lac operon
 

1. To review LAC Operon

2. To continue with the pGlo Lab

a) predict what out test plates will look like.

b) Make observations on from your lab results

c) complete packet questions.

                                                                                                                                   Correction on RNA polymerase II :  12 subunits = 12 different exons on the same transcription unit. I previous may have stated that this structure was quaternary.                                                                          

  _______________________________

Lac Operon Notes: 

 The Lac Operon is segment on the bacterial DNA includes a single promoter for a group of genes.  These genes represent the 3 enzymes in a single metabolic pathway that will digest Lactose.  This is a catabolic operon as it breaks down a larger molecules (disaccharide)  into a smaller molecule it can metabolize for energy (glucose).  It is an inducible operon that is the “off” position until a repressor protein produced from an upstream regulatory gene is induced by allolactose to allosterically change its shape so that it can no longer bind  to the operator and thus will turn the operon “on” when there is lactose present in the cell.

It is a negative control and positive control operon.


                         Permease             —–>            B – Galactosidase – 

              aids in the absorption of Lactose in the cell                                    Hydrolyzes the 1,4 glycosidic bond in Lactose

                                   Currently is is unclear what the role of Transacetylase is.

                                                                                                                                                                  –                                                                                                                                                                                                                 

Lac Operon:

  _______________________________

pGlo Plasmid: 

pGlo data: Morgan and Christie 2019

-pGlo – LB                                                     

E. Coli that DID NOT HAVE THE pGlO plasmid cultured on the original LB agarose medium.  Bacteria grew as a biofilm instead of colonies as a result of too much of the E. Coli suspension added

Proved Bacteria were viable.

__________________________________________

+pGlo – LB/Amp

E.Coli that DID HAVE THE pGlo plasmid cultured on the  LB agarose medium WITH AMPICILLIN. Bacteria grew as colonies.
Proved that all bacteria were transformed due to
Ampicillin resistance.

– pGlo – LB/AMP

E.Coli that DID NOT HAVE THE pGlO plasmid cultured  on LB agarose medium WITH AMPICILLIN.

There was no Bacteria colonies or biofilm.  

Proved non transformed bacteria do not haver ampicillin resistance. 

_____________________________________

 +pGlo – LB/Amp/Ara

E.Coli that did have the pGlo plasmid cultured on the LB ararose mediun WITH AMPICILLIN and AGAROSE.  Bacteria had the Green Glowing allele.

Proved that agarose regulates the pGlo gene.

 

 

Drew, David, Nick (and Luke?) 2021:

 

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12/22 – Wednesday’s Homework: –

                                                                       

1:  Please answer questions in PGlo lab packet page 3 (questions 1 – 4) and
     page 12 (questions 1 – 4) , page 13, page 16 (questions 1,2,3)
     This lab Packet is due this Tomorrow!                                                                                                            
2.   boys husky, extra tall, just saying… 
 

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12/23 – Thursday –  B Day 

Main focus –                                                                                                                                                          
a)  To complete AP problems on transformation experiments
b)  To isolate DNA.
c)  Begin 
  
1.  AP Question practice on Transformation experiments.   
                                                                                                                                                                                              2. Start our fly experiment – Sex linked Fly lab.
  
3.  DNA Holiday Lab – Isolating DNA
     
                                                                                                                                                                                                          

_____________________

12/23 – Holiday Break Homework: –

1. Please check countdown everyday and fill in the form below with the exact time left to prove your              diligence!

 
1: Holiday Form:

End  of Tuesday…

 

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12/18 – Friday –  Christmas EveHoliday Break!

         
                                                                               See you next year!